Auxin Metabolome Profiling in the Arabidopsis Endoplasmic Reticulum Using an Optimised Organelle Isolation Protocol

The endoplasmic reticulum (ER) is an extensive network of intracellular membranes. Its major functions include proteosynthesis, protein folding, post-transcriptional modification and sorting of proteins within the cell, and lipid anabolism. Moreover, several studies have suggested that it may be involved in regulating intracellular auxin homeostasis in plants by modulating its metabolism. Therefore, to study auxin metabolome in the ER, it is necessary to obtain a highly enriched (ideally, pure) ER fraction. Isolation of the ER is challenging because its biochemical properties are very similar to those of other cellular endomembranes. Most published protocols for ER isolation use density gradient ultracentrifugation, despite its suboptimal resolving power. Here we present an optimised protocol for ER isolation from Arabidopsis thaliana seedlings for the subsequent mass spectrometric determination of ER-specific auxin metabolite profiles. Auxin metabolite analysis revealed highly elevated levels of active auxin form (IAA) within the ER compared to whole plants. Moreover, samples prepared using our optimised isolation ER protocol are amenable to analysis using various “omics” technologies including analyses of both macromolecular and low molecular weight compounds from the same sample.

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Transmission Electron Microscopy of Protein Macromolecules

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100066176 ◽

1971 ◽

Vol 29 ◽

pp. 424-425

Author(s):

Henry S. Slayter

Keyword(s):

Biological Systems ◽

Metal Vapor ◽

Resolving Power ◽

Electron Microscopic ◽

Chemical Methods ◽

Molecular Weights ◽

The Past ◽

Physical Chemical ◽

Transmission Electron

Electron microscopic methods have been applied increasingly during the past fifteen years, to problems in structural molecular biology. Used in conjunction with physical chemical methods and/or Fourier methods of analysis, they constitute powerful tools for determining sizes, shapes and modes of aggregation of biopolymers with molecular weights greater than 50, 000. However, the application of the e.m. to the determination of very fine structure approaching the limit of instrumental resolving power in biological systems has not been productive, due to various difficulties such as the destructive effects of dehydration, damage to the specimen by the electron beam, and lack of adequate and specific contrast. One of the most satisfactory methods for contrasting individual macromolecules involves the deposition of heavy metal vapor upon the specimen. We have investigated this process, and present here what we believe to be the more important considerations for optimizing it. Results of the application of these methods to several biological systems including muscle proteins, fibrinogen, ribosomes and chromatin will be discussed.

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Experimental Determination of the Effective Resolution Limit in Thick Specimens at High Voltages

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100116942 ◽

1975 ◽

Vol 33 ◽

pp. 664-665

Author(s):

Mircea Fotino

Keyword(s):

Experimental Approach ◽

Chromatic Aberration ◽

Resolving Power ◽

Biological Research ◽

Specimen Thickness ◽

Resolution Limit ◽

High Voltage Electron Microscopy ◽

Voltage Electron ◽

Resolution Level

The use of thick specimens (0.5 μm to 5.0 μm or more) is one of the most resourceful applications of high-voltage electron microscopy in biological research. However, the energy loss experienced by the electron beam in the specimen results in chromatic aberration and thus in a deterioration of the effective resolving power. This sets a limit to the maximum usable specimen thickness when investigating structures requiring a certain resolution level.An experimental approach is here described in which the deterioration of the resolving power as a function of specimen thickness is determined. In a manner similar to the Rayleigh criterion in which two image points are considered resolved at the resolution limit when their profiles overlap such that the minimum of one coincides with the maximum of the other, the resolution attainable in thick sections can be measured by the distance from minimum to maximum (or, equivalently, from 10% to 90% maximum) of the broadened profile of a well-defined step-like object placed on the specimen.

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Biochemical study of attached macroalgae from the Madeira Archipelago and beach-cast macroalgae from the Canary Islands: multivariate analysis to determine bioresource potential

Botanica Marina ◽

10.1515/bot-2019-0022 ◽

2020 ◽

Vol 63 (3) ◽

pp. 283-298

Author(s):

Nuno Nunes ◽

Sofia Valente ◽

Sónia Ferraz ◽

Maria Carmo Barreto ◽

Miguel A.A. Pinheiro de Carvalho

Keyword(s):

Biochemical Study ◽

Algal Species ◽

Biochemical Properties ◽

Total Phenolic ◽

Total Carotenoids ◽

The North ◽

Madeira Archipelago ◽

Total Carbohydrates ◽

Beach Cast

AbstractFifteen attached macroalgae from the Madeira Archipelago, comprising three green, three red and nine brown algal species, as well as two beach-cast macroalgal samples, collected along the north shore of Gran Canaria, were assessed for their biochemical properties. The analysis included the determination of total minerals, total carbohydrates, protein, lipids, chlorophyll a, total carotenoids, total phenolic content, fucoxanthin and phycobilins (allophycocyanin, phycocyanin and phycoerythrin). The results showed a high variability of biochemical composition, allowing for the targetting of specific bioresources for particular purposes, including functional foods. This work provides the foundation for a biorefinery strategy implementation plan, for which specific macroalgae may be targeted for valuable and beneficial compounds.

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Simultaneous Determination of Human Serum Albumin and Low-Molecular-Weight Thiols after Derivatization with Monobromobimane

Molecules ◽

10.3390/molecules26113321 ◽

2021 ◽

Vol 26 (11) ◽

pp. 3321

Author(s):

Katarzyna Kurpet ◽

Rafał Głowacki ◽

Grażyna Chwatko

Keyword(s):

Molecular Weight ◽

Human Serum Albumin ◽

Serum Albumin ◽

Human Serum ◽

Simultaneous Determination ◽

Reversed Phase ◽

Fluorescence Labeling ◽

Detector Response ◽

Low Molecular Weight

Biothiols are extremely powerful antioxidants that protect cells against the effects of oxidative stress. They are also considered relevant disease biomarkers, specifically risk factors for cardiovascular disease. In this paper, a new procedure for the simultaneous determination of human serum albumin and low-molecular-weight thiols in plasma is described. The method is based on the pre-column derivatization of analytes with a thiol-specific fluorescence labeling reagent, monobromobimane, followed by separation and quantification through reversed-phase high-performance liquid chromatography with fluorescence detection (excitation, 378 nm; emission, 492 nm). Prior to the derivatization step, the oxidized thiols are converted to their reduced forms by reductive cleavage with sodium borohydride. Linearity in the detector response for total thiols was observed in the following ranges: 1.76–30.0 mg mL−1 for human serum albumin, 0.29–5.0 nmol mL−1 for α-lipoic acid, 1.16–35 nmol mL−1 for glutathione, 9.83–450.0 nmol mL−1 for cysteine, 0.55–40.0 nmol mL−1 for homocysteine, 0.34–50.0 nmol mL−1 for N-acetyl-L-cysteine, and 1.45–45.0 nmol mL−1 for cysteinylglycine. Recovery values of 85.16–119.48% were recorded for all the analytes. The developed method is sensitive, repeatable, and linear within the expected ranges of total thiols. The devised procedure can be applied to plasma samples to monitor biochemical processes in various pathophysiological states.

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Exploring Secondary Metabolite Profiles of Stachybotrys spp. by LC-MS/MS

Toxins ◽

10.3390/toxins11030133 ◽

2019 ◽

Vol 11 (3) ◽

pp. 133 ◽

Cited By ~ 3

Author(s):

Annika Jagels ◽

Viktoria Lindemann ◽

Sebastian Ulrich ◽

Christoph Gottschalk ◽

Benedikt Cramer ◽

...

Keyword(s):

Secondary Metabolites ◽

Future Research ◽

Structural Modifications ◽

Metabolite Profiles ◽

The Individual ◽

First Time ◽

Analytical Standards ◽

Respective Species

The genus Stachybotrys produces a broad diversity of secondary metabolites, including macrocyclic trichothecenes, atranones, and phenylspirodrimanes. Although the class of the phenylspirodrimanes is the major one and consists of a multitude of metabolites bearing various structural modifications, few investigations have been carried out. Thus, the presented study deals with the quantitative determination of several secondary metabolites produced by distinct Stachybotrys species for comparison of their metabolite profiles. For that purpose, 15 of the primarily produced secondary metabolites were isolated from fungal cultures and structurally characterized in order to be used as analytical standards for the development of an LC-MS/MS multimethod. The developed method was applied to the analysis of micro-scale extracts from 5 different Stachybotrys strains, which were cultured on different media. In that process, spontaneous dialdehyde/lactone isomerization was observed for some of the isolated secondary metabolites, and novel stachybotrychromenes were quantitatively investigated for the first time. The metabolite profiles of Stachybotrys species are considerably influenced by time of growth and substrate availability, as well as the individual biosynthetic potential of the respective species. Regarding the reported adverse effects associated with Stachybotrys growth in building environments, combinatory effects of the investigated secondary metabolites should be addressed and the role of the phenylspirodrimanes re-evaluated in future research.

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Proteomic Determination of Low-Molecular-Weight Glutenin Subunit Composition in Aroona Near-Isogenic Lines and Standard Wheat Cultivars

International Journal of Molecular Sciences ◽

10.3390/ijms22147709 ◽

2021 ◽

Vol 22 (14) ◽

pp. 7709

Author(s):

Kyoungwon Cho ◽

You-Ran Jang ◽

Sun-Hyung Lim ◽

Susan B. Altenbach ◽

Yong Q. Gu ◽

...

Keyword(s):

Molecular Weight ◽

Allelic Variation ◽

Reversed Phase ◽

Glutenin Subunit ◽

Low Molecular Weight ◽

Near Isogenic Lines ◽

Wheat Cultivars ◽

Reliable Determination ◽

Isogenic Lines

The low-molecular weight glutenin subunit (LMW-GS) composition of wheat (Triticum aestivum) flour has important effects on end-use quality. However, assessing the contributions of each LMW-GS to flour quality remains challenging because of the complex LMW-GS composition and allelic variation among wheat cultivars. Therefore, accurate and reliable determination of LMW-GS alleles in germplasm remains an important challenge for wheat breeding. In this study, we used an optimized reversed-phase HPLC method and proteomics approach comprising 2-D gels coupled with liquid chromatography–tandem mass spectrometry (MS/MS) to discriminate individual LMW-GSs corresponding to alleles encoded by the Glu-A3, Glu-B3, and Glu-D3 loci in the ‘Aroona’ cultivar and 12 ‘Aroona’ near-isogenic lines (ARILs), which contain unique LMW-GS alleles in the same genetic background. The LMW-GS separation patterns for ‘Aroona’ and ARILs on chromatograms and 2-D gels were consistent with those from a set of 10 standard wheat cultivars for Glu-3. Furthermore, 12 previously uncharacterized spots in ‘Aroona’ and ARILs were excised from 2-D gels, digested with chymotrypsin, and subjected to MS/MS. We identified their gene haplotypes and created a 2-D gel map of LMW-GS alleles in the germplasm for breeding and screening for desirable LMW-GS alleles for wheat quality improvement.

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Analytical subcellular fractionation of needle-biopsy specimens from human liver

Biochemical Journal ◽

10.1042/bj1740435 ◽

1978 ◽

Vol 174 (2) ◽

pp. 435-446 ◽

Cited By ~ 39

Author(s):

T J Peters ◽

C A Seymour

Keyword(s):

Endoplasmic Reticulum ◽

Plasma Membrane ◽

Density Gradient ◽

Needle Biopsy ◽

Human Liver ◽

Resolving Power ◽

Equilibrium Density ◽

Marker Enzyme ◽

Acid Hydrolases ◽

Biopsy Specimens

1. Fragments (2-20 mg wet wt.) of closed needle-biopsy specimens from human liver were disrupted in iso-osmotic sucrose and subjected to low-speed centrifugation. The supernatant was layered on a linear sucrose-density gradient in the Beaufay small-volume automatic zonal rotor. The following organelles, with equilibrium densities (g/ml) and principal marker enzyme shown in parentheses, were resolved: plasma membrane (1.12-1.14; 5′-nucleotidase); lysosomes (1.15-1.20; N-acetyl-beta-glucosaminidase); mitochondria (1.20; malate dehydrogenase); endoplasmic reticulum (1.17-1.21; neutral alpha-glucosidase); peroxisomes (1.22-1.24; catalase). 2. The distribution of particulate alkaline phosphatase and, to a lesser degree, leucine 2-naphthylamidase followed that of 5′-nucleotidase. gamma-Glutamyltransferase was associated with membranes of significantly higher equilibrium density than was 5′-nucleotidase. 3. The distribution of 12 acid hydrolases was determined in the density-gradient fractions. beta-Glucosidase had a predominantly cytosolic localization, but the other enzymes showed a broad distribution of activity throughout the gradient. Evidence was presented for two populations of lysosomes with equilibrium densities of 1.15 and 1.20 g/ml, but containing differing amounts of each enzyme. Further evidence of lysosomal heterogeneity was demonstrated by studying the distribution of isoenzymes of hexosaminidase and of acid phosphatase. 4. The resolving power of the centrifugation procedure can be further enhanced with membrane perturbants. Digitonin (0.12 mM) selectively disrupted lysosomes, markedly increased the equilibrium density of plasma-membrane components and lowered the density of the endoplasmic reticulum, but did not affect the mitochondria or peroxisomes. Pyrophosphate (15 mM) selectively lowered the equilibrium density of the endoplasmic reticulum.

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