Phenotyping hepatocellular metabolism using uniformly labeled carbon-13 molecular probes and LC-HRMS stable isotope tracing

Metabolite stable isotope tracing is a powerful bioanalytical strategy that has the potential to unravel phenotypic markers of early pharmaceutical efficacy by monitoring enzymatic incorporation of carbon-13 atoms into targeted pathways over time. The practice of probing biological systems with carbon-13 labeled molecules using broad MS-based screens has been utilized for many years in academic laboratories but has had limited application in the pharmaceutical R&D environment. The goal of this work was to establish a LCMS analytical workflow that was capable of monitoring carbon-13 isotope changes in glycolysis, the TCA and urea cycles, and non-essential amino acid metabolism. This work applies a standardized protein precipitation with 80% cold methanol and two distinct reverse-phase ion-pair liquid chromatography methods coupled to either a positive- or negative-ion mode high-resolution accurate mass spectrometry screening method. The data herein combines thousands of single-point peak integrations into a novel metabolite network map as a visualization aid to probe and monitor stable isotope incorporation in murine hepatocytes using uniformly labeled ¹³C₆ glucose, ¹³C₃ lactate, and ¹³C₅ glutamine. This work also demonstrates that nitrogen metabolism may have a large influence on the TCA cycle and gluconeogenic carbon fluxes in hepatocyte cell culture.     

Alcohol interactions with lipids: a carbon-13 nuclear magnetic resonance study using butanol labeled at C-1

The interactions of carbon-13 enriched butanol with dipalmitoylphosphatidylcholine (DPPC) and dimyristoylphosphatidylcholine (DMPC) were studied using C-13 nuclear magnetic resonance. It was found that above the gel to liquid crystal phase transition the resonance from the butanol could be resolved into two signals with similar chemical shifts but different T1 values and line widths. In contrast, only one narrow resonance was observed for ethanol, which has considerably less solubility in the lipids than butanol. Thermodynamic analyses of the effects of butanol on the phase transition temperature predict much greater solubility or butanol when the lipid is above the phase transition temperature than when it is below. It was concluded that the two butanol resonances represent two slowly exchanging populations, the free butanol in the aqueous phase and butanol dissolved in the liquid crystalline region of the lipid. No bound butanol was detected below the gel to liquid crystal phase transition. Relaxation studies were performed on the resonance of the bound butanol in DPPC and DMPC, including measurements of T1, line width, and nuclear Overhauser enhancement. Theoretical analysis of the relaxation parameters indicates that the lipid-bound alcohol has very high mobility within the fluid lipid bilayer. The data are consistent with butanol being present at the aqueous boundary or head group region of the lipid.     

Effects of leaf soluble sugars content and net photosynthetic rate of quince donor shoots on subsequent morphogenesis in leaf explants

The effects of different growth conditions (ventilated and closed vessels, medium with 0, 15 and 30 g dm⁻³ sucrose) during proliferation of donor quince (Cydonia oblonga Mill.) shoots (stage I) on net photosynthetic rate and soluble sugars content were evaluated. In order to assess the influence of these physiological parameters on morphogenesis, leaf explants harvested from donor shoots were induced to form somatic embryos and adventitious roots under ventilated and closed Petri dishes (stage II). Natural ventilation and low sucrose contents (0–15 g dm⁻³) promoted the photosynthetic rate of quince shoots whereas biomass accumulation was the highest in those shoots cultured with 30 g dm⁻³ sucrose in both vessel types and 15 g dm⁻³ sucrose under natural ventilation. Increasing sucrose content in the medium induced greater accumulation of sucrose in leaf tissues of donor shoots. The content of reducing sugars was higher than that of sucrose, and it appeared to be higher in shoots cultured under natural ventilation compared to those in closed vessels. Somatic embryogenesis and root regeneration were influenced by stage I and II treatments. A significant correlation between sucrose content in the leaves of donor shoots and the number of somatic embryos regenerated was found, suggesting that identification of biochemical and physiological characteristics of donor shoots associated with increased regeneration ability might be helpful for improving morphogenesis in plant tissue culture.     

Large-scale phenotyping of transgenic tobacco plants (Nicotiana tabacum) to identify essential leaf functions

Two of the major challenges in functional genomics are to identify genes that play a key role in biological processes, and to elucidate the biological role of the large numbers of genes whose function is poorly characterized or still completely unknown. In this study, a combination of large-scale expressed sequence tag sequencing, high-throughput gene silencing and visual phenotyping was used to identify genes in which partial inhibition of expression leads to marked phenotypic changes, mostly on leaves. Three normalized tobacco (Nicotiana tabacum) cDNA libraries were prepared directly in a binary vector using different tissues of tobacco as an RNA source, randomly sequenced and clustered. The Agrobacterium-tobacco leaf disc transformation system was used to generate sets of antisense or co-suppression transgenic tobacco plants for over 20 000 randomly chosen clones, each representing an independent cluster. After transfer to the glasshouse, transgenic plants were scored visually after 10-14 days for changes in growth, leaf form and chlorosis or necrosis. Putative hits were validated by repeating the transformation. This procedure is more stringent than the analysis of knockout mutants, because it requires that even a partial decrease in expression generates a phenotype. This procedure identified 88 validated gene/phenotype relations. These included several previously characterized gene/phenotype relationships, demonstrating the validity of the approach. For about one-third, a function could be inferred, but a loss-of-function phenotype had not been described previously. Strikingly, almost one-half of the validated genes were poorly annotated, or had no known function. For 77 of these tobacco sequences, a single or small number of potential orthologues were identified in Arabidopsis. The genes for which orthologues were identified in Arabidopsis included about one-half of the genes whose function was completely unknown. Comparison with published gene/phenotype relations for Arabidopsis knockout mutants revealed surprisingly little overlap with the present study. Our results indicate that partial gene silencing identifies novel gene/phenotype relationships, which are distinct from those uncovered by knockout screens. They also show that it is possible to perform these analyses in a crop species in which full genome sequence information is lacking, and subsequently to transfer the information to a reference species in which functional studies can be performed more effectively.     

Enhancement by ethylene of cellulysin-induced ethylene production by tobacco leaf discs

Cellulysin-induced ethylene production in tobacco (Nicotiana tabacum L.) leaf discs was enhanced several-fold by prior exposure of the leaf tissue to ethylene. This enhancement in the response of the tissue to Cellulysin increased rapidly during 4 and 8 hours of pretreatment with ethylene and resulted from greater conversion of methionine to ethylene. On treatment with Cellulysin, the content of 1-aminocyclopropane-1-carboxylic acid (ACC) in leaf discs not pretreated with ethylene markedly increased while that of the ethylene-pretreated tissue was only slightly higher than in the tissue incubated in the absence of Cellulysin. Ethylene-treated tissue, however, converted ACC to ethylene at a faster rate than air controls. These data indicate that ethylene stimulates Cellulysin-induced ethylene production by stimulating the conversion of ACC to ethylene. Data are also presented on a possible relation of this phenomenon to ethylene produced by the tobacco leaf upon interaction with its pathogen, Alternaria alternata.     

Carbon-13 NMR studies of chloroplast membranes: carbon-13 enrichment without 13C-13C couplings

A novel approach to carbon-13 (13C) enrichment of chloroplast membranes (and plant materials in general) is presented for 13C-nuclear magnetic resonance (13C-NMR) studies. The method minimizes the occurrence of spectral complications arising from 13C-13C couplings resulting from a statistical distribution of 13C within the molecule with low probability of encountering two 13C atoms adjacent to each other. This is achieved by growing the plants in light surrounded by an atmosphere containing 1/3rd 12CO2 and 2/3rd 13CO2, liberated by weak acid-treatment of a mixture of corresponding barium carbonate salts.     

Role of IAA conjugates in inducing ethylene production by tobacco leaf discs

Indole-3-acetic acid (IAA) labeled in its carboxyl group was metabolized by tobacco leaf discs (Nicotiana tabacum L. cv. Xanthi) into three metabolites, two of which were preliminarily characterized as a peptide and an ester-conjugated IAA. Reapplication of each of the three metabolites (at 10 μM) resulted in a marked stimulation of ethylene production and decarboxylation by the leaf discs. Similarly, these three IAA metab olites could induce elongation of wheat coleoptile segments, which was accompanied by decarboxylation. Both the exogenously supplied esteric and peptidic IAA conjugates were converted by the leaf discs into the same metabolites as free IAA. (1-¹⁴C)IAA, applied to an isolated epidermis tissue, was completely metabolized to the esteric and peptidic IAA conjugates. This epidermis tissue showed much higher ethylene production rates and lower decarboxylation rates than did the whole leaf disc. The results suggest that the participation of IAA conjugates in the regulation of various physiological processes depends on the release of free IAA, which is obtained by enzymatic hydrolysis of the conjugates in the tissue. The present study demonstrates biological activity of endogenous IAA conjugates that were synthesized by tobacco leaf discs in response to exogenously supplied IAA.     

Assessment of the production of 13C labeled compounds from phototrophic microalgae at laboratory scale

An integrated process for the indoor production of 13C labeled polyunsaturated fatty acids (PUFAs) from Phaeodactylum tricornutum is presented. The core of the process is a bubble column photobioreactor operating with recirculation of the exhaust gas using a low-pressure compressor. Oxygen accumulation in the system is avoided by bubbling the exhaust gas from the reactor in a sodium sulfite solution before returning to it. To achieve a high 13C enrichment in the biomass obtained, the culture medium is initially stripped of carbon, and labeled 13CO(2) is automatically injected on-demand during operation for pH control and carbon supply. The reactor was operated in both batch and semicontinuous modes. In semicontinuous mode, the reactor was operated at a dilution rate of 0.01 h(-1), resulting in a biomass productivity of 0.1 g l(-1) per day. The elemental analysis of the inlet and outlet flows of the reactor showed that 64.9% of carbon was turned into microalgal biomass, 34.9% remained in the supernatant mainly as inorganic compounds. Only 3.8% of injected carbon was effectively fixed as the target labeled product (EPA). Regarding the isotopic composition of fatty acids, results showed that fatty acids were not labeled in the same proportion, the higher the number of carbons the lower the percentage of 13C. Isotopic composition of EPA ranged from 36.5 to 53.5%, as a function of the methodology used (GC-MS, EA-IRMS or gas chromatography-combustion-isotope ratio mass spectrometry (GC-IRMS)). The low carbon uptake efficiency combined with the high cost of 13CO(2) make necessary to redefine the designed culture system to increase the efficiency of the conversion of 13CO(2) into the target product. Therefore, the possibility of removing 12C from the fresh medium, and recovering and recirculating the inorganic carbon in the supernatant and the organic carbon from the EPA depleted biomass was studied. The inorganic carbon of the fresh medium was removed by acidification and stripping with N(2). The inorganic carbon of the supernatant was recovered also by acidification and subsequent stripping with N(2). The operating conditions of this step were optimized for gas flow rate and type of contactor. A carbon recovery step for the depleted biomass was designed based on the catalytic oxidation to CO(2) using CuO (10 wt.%) as catalyst with an oxygen enriched atmosphere (80% O(2) partial pressure). In this way, the carbon losses reduced an 80.2% and the efficiency of the conversion of carbon in EPA was increased to 19.5%, which is close to the theoretical maximum. Further increase in 13CO(2) use efficiency is only possible by additionally recovering other labeled by-products present in the biomass: proteins, carbohydrates, lipids, and pigments.     

Large-scale preparation of asymmetrically labeled fluorescent lipid vesicles

A method for producing lipid vesicles containing fluorescent phospholipid analogues localized to the inner leaflet of their membrane was developed. Incubation of a 450-fold molar excess of serum albumin with lipid vesicles symmetrically labeled with 1 mol % 1-palmitoyl-2-(N-4-nitrobenzo-2-oxa-1,3-diazolyl)amino-caproyl phosphatidylcholine resulted in the removal of 99% of the fluorescent lipid from the outer leaflet. Asymmetrically labeled vesicles were separated from albumin/lipid complexes by gel filtration chromatography. Vesicles prepared in this manner were unable to transfer fluorescent lipid to cells during liposome-cell incubations. Liposomes asymmetrically labeled with other 4-nitrobenzo-2-oxa-1,3-diazole (NBD)-phospholipid analogues were also prepared. Removal of amino-dodecanoyl-NBD-labeled lipids from the outer leaflet of liposomes required three times more bovine serum albumin, and 48 h of incubation. This method can be used to produce large amounts of asymmetrically labeled liposomes suitable for use in investigating a variety of membrane phenomena.     

Plant photosynthetic production as controlled by leaf growth,phenology, and behavior

In this historical review we summarize discoveries related to the flowering genes in controlling leaf area index (LAI, the leaf area per unit ground area) in sorghum, soybean, or pea crop stands. We also analyze similar work on Arabidopsis and dwarf and intermediate stem height genes in wheat and rice.     

Use of carbon-13 and carbon-14 natural abundances for stream food web studies

We review the use of stable carbon isotope ratios (δ¹³C) and radiocarbon natural abundances (Δ¹⁴C) for stream food web studies. The δ¹³C value of primary producers (e.g., periphytic algae, hereafter periphyton) in streams is controlled by isotopic fractionation during photosynthesis and variable δ¹³C of dissolved CO₂. When periphyton δ¹³C differs from that of terrestrial primary producers, the relative contribution of autochthony and allochthony to stream food webs can be calculated. Moreover, the variation in periphyton δ¹³C can reveal how much stream consumers rely on local resources because each stream habitat (e.g., riffle vs. pool, open vs. shaded) usually has a distinctive δ¹³C. However, periphyton δ¹³C often overlaps with that of terrestrial organic matter. On the other hand, periphyton Δ¹⁴C is less variable than δ¹³C among habitats, and reflects the Δ¹⁴C of dissolved CO₂, which could be a mixture of “aged” (Δ¹⁴C < 0 ‰) and “modern” (Δ¹⁴C > 0 ‰) carbon. This is because the Δ¹⁴C is corrected by its δ¹³C value for the isotopic fractionation during photosynthesis. Recent studies and our data indicate that many stream food webs are supported by “aged” carbon derived from the watershed via autochthonous production. The combined use of δ¹³C and Δ¹⁴C allows robust estimation of the carbon transfer pathway in a stream food web at multiple spatial scales ranging from the stream habitat level (e.g., riffle and pool) to watershed level (autochthony and allochthony). Furthermore, the Δ¹⁴C of stream food webs will expand our understanding about the time frame of carbon cycles in the watersheds.     

Investigation of the interactions of oxytocin with neurophysins at low pH using carbon-13 nuclear magnetic resonance and carbon-13-labeled hormones.

The specifically 13C-labeled (90% 13C-enriched) peptide hormone derivatives [1-hem[2-13C]cystine]oxytocin, [1-hemi[1-13C]cystine]oxytocin, and [2-[-2-13C]tyrosine[-oxytocin and the analogue [3-[2-13C]leucine]oxytocin were prepared by total synthesis and used to study the interactions of the neurohypophyseal hormones with the bovine neurophysins as a function of pH and temperature. Under all conditions, whether high or low pH, the chemical shifts of the labeled carbon atoms of the bound hormones are the same, but they are shifted significantly from their positions in the free hormone. These results indicate that interactions of the side chain and disulfide moieties of the hormone with the neurophysins do not change as a function of pH. At neutral pH and 20--35 degrees C, the labeled atoms of the hormone are in slow exchange (1--5 s-1) with the neurophysins for the above hormone derivatives, but at low pH they are in intermediate or fast exchange depending upon the pH and temperature. At low pH, the dissociation rate constant (koff) is about 100-fold greater than the value at neutral pH, and this increase appears to be due exclusively to the breaking of the salt bridge involving the N-terminal amino group of oxytocin and a side-chain carboxyl group of neurophysin. Since the dissociation constant (Kd) also increases by about 100-fold in going from neutral to low pH, the association rate constant is deduced to be the same at neutral and low pH. In contrast to the low pH results, an increase in pH (from 6.6 to 10.5) leads to a continual decrease in the binding constant but to no apparent change in the dissociation rate constant. The bound hormone is always in slow exchange at high pH, even when the binding constant has been reduced by 2 or 3 orders of magnitude. At high pH, the decrease in binding affinity is due solely to the deprotonation of the alpha-amino group of the free hormone. Thus, at high pH the apparent association rate constant decreases, while the dissociation rate constant remains unchanged.     

A carbon-13 nuclear magnetic resonance study of the 3'-terminus of 16S ribosomal RNA of Escherichia coli specifically labeled with carbon-13 in the methylgroups of the m6(2)Am6(2)A sequence.

30S ribosomes were isolated from a kasugamycin resistant mutant of E. coli that lacks methylgroups on two adjacent adenines in 16S ribosomal RNA. These ribosomes were methylated in vitro with a purified methylating enzyme and 5-S-adenosyl-(13C-methyl)-L-methionine chloride ((13C-methyl)-SAM) as methyldonor. After in situ cleavage of the 16S ribosomal RNA by the bacteriocin cloacin DF13, the 49 nucleotide fragment from the 3'-end of the RNA was isolated. The carbon-13 nuclear magnetic resonance spectra of the fragment at various temperatures were compared with those of 6-N-dimethyladenosine (m6(2)A) and 6-N-dimethyladenylyl-(3' leads to 5')-6-N-dimethyladenosine (m6(2)Am6(2)A). The data show that the two methylated adenines, which are part of a four membered hairpin loop, show a strong tendency to be stacked in analogy to the dinucleotide m6(2)Am6(2).     

Carbon-13 NMR spectra of carbon-13 enriched chlorophylls a and b

The proton decoupled ¹³C NMR (CMR) spectra of chlorophylls a and b enriched to 90% ¹³C have been obtained at 25.2 MHz and, despite the complexity of the spectra, many of the assignments of the ¹³C resonances have been made.