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Pyrroline

Q: What are some common applications of Pyrroline?

Pyrrolines have a wide range of applications in various fields. They are used in the synthesis of pharmaceuticals, as building blocks for organic compounds, and in the development of new materials with unique properties. Pyrrolines are also important in biochemistry, as they are found in many natural compounds and play a role in biological processes.

Pyrroline is a heterocyclic organic compound containing a five-membered ring with four carbon atoms and one nitrogen atom.
It is a fundamental structural unit found in various biological molecules and compounds, including proline, the amino acid.
Pyrrolines exhibit diverse chemical and biological properties, making them important in fields such as medicinal chemistry, biochemistry, and materials science.
Researchers can utilize PubCompare.ai's innovative AI-powered platform to efficiently locate the most effective and reproducible protocls for working with pyrrolines across published literature, pre-prints, and patents, saveing time and enhancing research outcomes.

Most cited protocols related to «Pyrroline»

Detecting Apoptosis and Oxidative Stress in Yeast

Cited 42 times

Electron microscopy, annexin V labeling, and DAPI staining were performed as described previously (Madeo et al., 1997 (link)). For the TdT-mediated dUTP nick end labeling (TUNEL) test, cells were prepared as described (Madeo et al., 1997 (link)), and the DNA ends were labeled using the In Situ Cell Death Detection Kit, POD (Boehringer Mannheim). Yeast cells were fixed with 3.7% formaldehyde, digested with lyticase, and applied to a polylysine-coated slide as described for immunofluorescence (Adams and Pringle, 1984 (link)). The slides were rinsed with PBS and incubated with 0.3% H₂O₂ in methanol for 30 min at room temperature to block endogenous peroxidases. The slides were rinsed with PBS, incubated in permeabilization solution (0.1% Triton X-100 and 0.1% sodium citrate) for 2 min on ice, rinsed twice with PBS, incubated with 10 μl TUNEL reaction mixture (terminal deoxynucleotidyl transferase 200 U/ml, FITC-labeled dUTP 10 mM, 25 mM Tris-HCl, 200 mM sodium cacodylate, 5 mM cobalt chloride; Boehringer Mannheim) for 60 min at 37°C, and then rinsed 3× with PBS. For the detection of peroxidase, cells were incubated with 10 μl Converter-POD (anti-FITC antibody, Fab fragment from sheep, conjugated with horseradish peroxidase) for 30 min at 37°C, rinsed 3× with PBS, and then stained with DAB-substrate solution (Boehringer Mannheim) for 10 min at room temperature. A coverslip was mounted with a drop of Kaiser's glycerol gelatin (Merck). As staining intensity varies, only samples from the same slide were compared.
Free intracellular radicals were detected with dihydrorhodamine 123, dichlorodihydrofluorescein diacetate (dichlorofluorescin diacetate), or dihydroethidium (hydroethidine; Sigma Chemical Co.). Dihydrorhodamine 123 was added ad-5 μg per ml of cell culture from a 2.5-mg/ml stock solution in ethanol and cells were viewed without further processing through a rhodamine optical filter after a 2-h incubation. Dichlorodihydrofluorescein diacetate was added ad-10 μg per ml of cell culture from a 2.5 mg/ml stock solution in ethanol and cells were viewed through a fluorescein optical filter after a 2-h incubation. Dihydroethidium was added ad-5 μg per ml of cell culture from a 5 mg/ml aqueous stock solution and cells were viewed through a rhodamine optical filter after a 10-min incubation. For flow cytometric analysis, cells were incubated with dihydrorhodamine 123 for 2 h and analyzed using a FACS^® Calibur (Becton Dickinson) at low flow rate with excitation and emission settings of 488 and 525–550 nm (filter FL1), respectively.
Free spin trap reagents N-tert-butyl-α−phenylnitrone (PBN; Sigma-Aldrich) and 3,3,5,5,-tetramethyl-pyrroline N-oxide (TMPO; Sigma-Aldrich) were added directly to the cell cultures as 10-mg/ml aqueous stock solutions. Viability was determined as the portion of cell growing to visible colonies within 3 d.
To determine frequencies of morphological phenotypes (TUNEL, Annexin V, DAPI, dihydrorhodamine 123), at least 300 cells of three independent experiments were evaluated.

Madeo F., Fröhlich E., Ligr M., Grey M., Sigrist S.J., Wolf D.H, & Fröhlich K.U. (1999). Oxygen Stress: A Regulator of Apoptosis in Yeast. The Journal of Cell Biology, 145(4), 757-767.

Publication 1999

3,3,5,5-tetramethyl-1-pyrroline N-oxide Annexin A5 Antibodies, Anti-Idiotypic Cacodylate Cardiac Arrest Cell Culture Techniques Cell Death Cells cobaltous chloride DAPI deoxyuridine triphosphate dichlorofluorescin dihydroethidium dihydrorhodamine 123 DNA Nucleotidylexotransferase Domestic Sheep Electron Microscopy Ethanol Flow Cytometry Fluorescein Fluorescein-5-isothiocyanate Formaldehyde Free Radicals Gelatins Glycerin Horseradish Peroxidase hydroethidine Immunofluorescence Immunoglobulins, Fab In Situ Nick-End Labeling lyticase Methanol Oxides Peroxidase Peroxidases Peroxide, Hydrogen Phenotype Polylysine Protoplasm pyrroline Rhodamine Sodium Sodium Citrate TERT protein, human Triton X-100 Tromethamine Yeast, Dried

Amplex Red Assay for Oxidative Stress

Cited 13 times

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Publication 2012

Biological Assay Buffers Catalase Cattle Chelex 100 Filtration Ions Kidney Liver Metals Oxides Pentetic Acid Peroxidase Peroxide, Hydrogen Phosphates pyrroline Resins, Plant resorufin Superoxide Dismutase Transition Elements TRAP1 protein, human

Quantification of Proline, GABA, and 2-AP in Grains

Cited 7 times

For determination of proline, GABA, and 2-AP, fresh sample of grains from 1 m² area of each plot were harvested and immediately stored at −20°C. Duplicate measurements were taken for each sample and the mean value of the three samples per treatment was taken as the final result.
The proline content was determined according to the method described by Bates et al. (1973 (link)). Briefly, grains (about 0.3 g) was homogenized in 5 mL of 3% sulfosalicylic acid, then cooled after heating at boiling water bath for 10 min. Samples filtered and two ml of the filtrate was mixed with 3 mL of ninhydrin reagent ( 1.25 g ninhydrin in 30 mL glacial acetic acid and 20 mL 6 M phosphoric acid) and 2 mL of glacial acetic acid. The reaction mixture was then heated at boiling water bath for 30 min and placed in an ice bath for 20 min before being extracted with 4 ml of toluene. The toluene extraction was then centrifuged at 4000 rpm for 5 min. The absorbance of the red chromophore in the toluene fraction was measured at 520 nm and the amount of proline was determined by comparison with a standard curve and expressed as μg∙g⁻¹.
GABA content in grain was determined as described by Zhao et al. (2009 ) and Yao et al. (2008 ). Briefly, grain (about 0.5 g) was homogenized in 5 mL of 60% ethanol, treated for 4 hours in a oscillations instrument (HZS-H, China) using a frequency of 200 oscillations per minute. The supernatant was then transferred to a 5 ml centrifuge tube and centrifuged at 8000 rpm for 3 min. 1 mL of supernatant was added to a 10 mL tube, mixed with 0.6 mL 0.2 mol∙L⁻¹ (pH 9.0) sodium tetraborate, two mL 5% toluene, and 1 mL 7% sodium hypochlorite, then cooled after heating at 100°C in a water bath for 5 minutes. The absorbance of the reaction solution was measured at 645 nm and the amount of GABA was determined by comparison with a standard curve and expressed as μg∙g⁻¹.
Grain samples were evaluated for 2-AP concentration by synchronization distillation and extraction method (SDE) combined with GCMS-QP 2010 Plus (Shimadzu Corporation, Japan) as described by Huang et al. (2012 ). Briefly, Collidine (2, 4, 6-trimethylpyridine) (Sigma, Switzerland), was used as an internal standard. 10 g of finely ground grain was transferred into a 500 mL round-bottom flask containing 145 mL purified water; 5 mL of 0.914 μg∙mL⁻¹ internal standard was then added. A steam distillation continuous extraction head was attached to the flask, and the flask was heated at 150°C by an oil bath pot, ZKYY (Guangzhou, China). Attached to the other head of the steam distillation continuous extraction instrument, diethyl ether (35 mL) was used as the solvent in a 500 mL round-bottom flask, and the flask was heated at 42°C by a water bath pot, HH-2 (Jiangsu, China). During isolation, the steam distillation continuous extraction was maintained at 10°C by a cold water circulation machine, YKKY-LX-300 (Beijing, China). The isolation was performed for 35 min. The ether extract was then dried over sodium sulfate, filtered (0.22 μm filter paper, Shimadzu, Japan), and then directly used to measure 2-AP concentrations with the GCMS-QP 2010 Plus method as described in Huang et al., (2012 ). The GCMS-QP 2010 Plus working conditions were as followings: gas chromatograph equipped with a Restek Rxi-5 ms (Shimadzu, Japan) silica capillary column (30 m × 0. 32 mm × 0. 25 μm). The auto injector was AOC-20i, SPL1. High purity helium gas (99.999%, Guangzhou Gases Co., LTD, China) was the carrier gas at the flow rate of 2.0 mL∙min⁻¹. The temperature of the GC oven was 40°C (1 min), increased at 2°C∙min⁻¹ to 65°C and held at 65°C for 1 min, and then increased to 220°C at 10°C∙min⁻¹, and held at 220°C for 10 min. The ion source temperature was 200°C. Under these conditions, the retention time of 2-AP was 7.5 min. 2-AP content was expressed as μg∙kg⁻¹. The relative content of the aroma compounds were identified on the basis of their mass spectra by comparing the spectra with the records of the NIST library.

Mo Z., Li W., Pan S., Fitzgerald T.L., Xiao F., Tang Y., Wang Y., Duan M., Tian H, & Tang X. (2015). Shading during the grain filling period increases 2-acetyl-1-pyrroline content in fragrant rice. Rice, 8, 9.

Publication 2015

Acetic Acid ARID1A protein, human Bath Capillaries cDNA Library Cereals Cold Temperature Distillation Ethanol Ethyl Ether gamma-collidine gamma Aminobutyric Acid Gas Chromatography Gas Chromatography-Mass Spectrometry Head Helium isolation Mass Spectrometry Ninhydrin Phosphoric Acids Proline Retention (Psychology) Scents Silicon Dioxide sodium borate Sodium Hypochlorite sodium sulfate Solvents Steam Strains sulfosalicylic acid Toluene

Criteria for Classifying Inborn Errors of Metabolism

Cited 7 times

We first established specific criteria to classify a disorder as an IEM. These criteria are summarized in Table 1, and explained in detail here.
In the current nosology, we ascribe to the recent redefinition of an IEM as any condition that leads to a disruption of a metabolic pathway, irrespective of whether it is associated with abnormalities in biochemical laboratory tests. We not only include primary enzyme or transporter deficiencies or superactivities, but also chaperone deficiencies–such as DNAJC12 deficiency presenting with hyperphenylalaninemia–or deficiencies in transcription factors that can induce metabolic abnormalities, such as HCF1 deficiency with methylmalonic aciduria, GATA1 deficiency with excess uroporphyrin I excretion, or NRF2 superactivity with hypohomocysteinemia.
We define single entries for individual enzyme deficiencies regardless of severity. For example, we do not include three separate entries for Gaucher disease types 1, 2 and 3, as this classification is largely dependent on severity of the enzyme deficiency, and not on a different pathophysiology. Similarly, there is only one entry for lysosomal acid lipase deficiency, and not two separate entries for Wolman disease and cholesteryl ester storage disease. However, we provide the alternative nomenclature for these disorders in a separate column. On the other hand, single enzyme deficiencies are included as two separate entries when they lead to clearly delineated phenotypes, not strictly associated with severity. For example, GM1 gangliosidosis and Morquio syndrome type B are both caused by β-galactosidase deficiency, but the phenotype is clearly distinct. Similarly, a deficiency of acid ceramidase can lead to Farber disease (of variable severity), but also to a different phenotype of spinal muscular atrophy with progressive myoclonic epilepsy, with no known correlation between the degree of enzyme activity and the phenotypic presentation. In the case of ATP7A-related distal motor neuropathy, the clinical and metabolic findings are completely unrelated to those of Menkes disease or occipital horn syndrome, and in fact the neuropathy phenotype is caused by distinct variants leading to a distinct pathomechanism. Such situations generate separate entries for defects in the same protein.
Conditions that can have both autosomal recessive and autosomal dominant inheritance are included under the same entry as long as they share a similar mechanism of disease, as is the case with MCT1 deficiency, SCOT deficiency, 5-oxoprolinase deficiency, ISCU deficiency, or hereditary hyperekplexia type 1. However, in some disorders, the different pattern of inheritance is associated with a completely different phenotype; for example, biallelic variants in genes encoding the subunits of succinate dehydrogenase lead to a leukodystrophy, while heterozygous variants in the same genes lead to familial paragangliomas, the latter likely via inhibition of HIF prolyl hydroxylases.^{9 (link)} For some disorders, the line is somewhat blurry. For example, autosomal dominant GTP cyclohydrolase deficiency manifests as dopa-responsive dystonia without hyperphenylalaninemia, while the autosomal recessive form manifests with earlier onset of severe neurologic dysfunction and hyperphenylalaninemia due to a more severe and systemic tetrahydrobiopterin deficiency. Thus, the current separation of this enzymatic defect into two separate entries is maintained, based on different clinical and biochemical findings, and different management. However, intermediate phenotypes do exist, as patients have been described with biallelic variants in GCH1 but only exhibiting dopa-responsive extrapyramidal symptoms without hyperphenylalaninemia,^{10 (link),11 (link)} while some heterozygous carriers exhibit hyperphenylalaninemia when given a phenylalanine load but otherwise are asymptomatic.^{12 (link)} Similarly, autosomal dominant hypermethioninemia caused by dominant negative variants in MAT1A is given a separate entry from the autosomal recessive MAT I/III deficiency caused by loss-of-function variants. In the case of Δ-1-pyrroline-5-carboxylate synthase deficiency, a predominant spastic paraplegia phenotype can be seen both with autosomal dominant or recessive inheritance, while a phenotype that includes neurologic involvement and cutis laxa can also be inherited in either an autosomal dominant or recessive fashion. The dominant inheritance of either the cutis laxa phenotype or the spastic paraplegia phenotype is likely due to dominant negative variants. ^{13 (link),14} In such cases, we include two entries for the same enzyme deficiency based on the clinical phenotype (as opposed to four separate entries, based on both phenotype and inheritance pattern). Classifying X-linked disorders as dominant or recessive can be problematic given the fact that females can have manifestations from X-linked conditions classically defined as recessive. ^{15 (link),16} Nevertheless, we elected to include two separate entries for X-linked disorders that present with different phenotypes in heterozygote females as compared to hemizygote males. Examples of this include the X-linked recessive CK syndrome or X-linked dominant CHILD syndrome, both caused by variants in NSDHL, or the X-linked recessive MEND syndrome vs X-linked dominant chondrodysplasia punctata, both caused by variants in EBP.
Different variants in some proteins can lead to either deficiency or superactivity, with drastically different phenotypes. Examples include the monocarboxylate transporter 1, δ-aminolevulinic acid synthase, phosphoribosylpyrophosphate synthetase, and glucokinase. Additionally, different variants in the same protein can lead to disparate mechanisms of disease. For example loss-of-function variants in the FTL gene lead to hereditary L-ferritin deficiency, while gain-of-function variants in exon 4 that alter the reading frame disrupt protein folding and stability and lead to neuroferritinopathy. Additionally, variants in the 5′ non-coding iron-responsive element (IRE) of the gene lead to hyperferritinemia-cataracts syndrome. Such cases are included as separate entries based on the discrepant pathophysiology.
Another criterion used in the current nosology was to include entries based on the particular gene product involved, and not solely on the clinical phenotype. For example, we included separate entries for glycine encephalopathy caused by the deficiency of glycine decarboxylase or aminomethyltransferase. Similarly, defects in many different mitochondrial transfer RNAs can lead to the MELAS phenotype, while defects in multiple nuclear-encoded mitochondrial proteins as well as mitochondrial DNA genes can lead to Leigh syndrome. We include separate entries for involvement of each such gene/protein. Numerous biochemical phenotypes that do not cause clinical disease have been described. Pentosuria, one of the first IEMs described by Archibald Garrod during his Croonian lectures, is asymptomatic.¹⁷ Other IEMs classically considered “non-diseases” include histidinemia, hydroxyprolinemia, or cystathioninuria. In contrast, some biochemical phenotypes originally thought to be clinically significant have been called into question (short-chain acyl-CoA dehydrogenase and 3-methylcrotonyl-CoA carboxylase deficiencies). Finally, some IEMs originally considered not to be clinically relevant have subsequently been associated with a disease phenotype, such as pterin-4-α-carbinolamine dehydratase deficiency.^{18 (link),19 (link)} All of these conditions have been included in the current nosology, as the term inborn error of metabolism does not by necessity denote a disease. We have not included variants in metabolism that are ubiquitous in humans as compared to other species, for example, lack of L-gulonolactone oxidase or urate oxidase activities.
Whenever possible, entries are named according to the protein involved, unless it is impractical. For example, the name “iminoglycinuria” is well established, and to insist instead on the protein-based name “proton amino acid transporter deficiency with or without imino acid transporter deficiency” is not likely to be generally accepted. Similarly, the name Hartnup disorder is well established, and preferable to “B° neutral amino acid transporter 1 deficiency”.
It should be noted that many entities could be assigned to more than one group based on function and phenotype, a situation that is acknowledged when pertinent. For example, succinate dehydrogenase functions both in the Krebs cycle and as part of the mitochondrial respiratory chain. In such cases, the specific phenotype is included in the group most directly related to the disease mechanism. For example, fructose-1,6-bisphosphatase deficiency is included in the group of disorders of gluconeogenesis, while it is only cross-referenced in the group of disorders of fructose metabolism.
We have not included conditions that remain unpublished, are not verified, or unlikely to have a genetic etiology. These conditions are considered to be poorly characterized either because their molecular basis has not been elucidated, because only a few patients have been described in decades-old reports, or because only individual families have been reported. However, it is possible that these proposed IEMs will be validated in the future.
Finally, certain contiguous gene deletions that include genes encoding for enzymes often have a phenotype distinct from the isolated (single gene) defect. Some examples include the combined deletion of MAOA and MAOB,^{20 (link)–22 (link)} contiguous ABCD1/DXS1375E deletion syndrome (also known as CADDS),^{23 (link)} hypotonia-cystinuria syndrome,^{24 (link),25 (link)} or complex glycerol kinase deficiency. They are not included in the nosology.

Ferreira C.R., van Karnebeek C.D., Vockley J, & Blau N. (2018). A proposed nosology of inborn errors of metabolism. Genetics in medicine : official journal of the American College of Medical Genetics, 21(1), 102-106.

Publication 2018

Inhalation Exposure System and Characterization

Cited 6 times

Exposure System and Aerosol Characterization: An animal inhalation exposure system was designed to expose animals to either CB aerosols, O₃ gas, or a mixture of the two toxicants (Supplemental Fig. S1). The design utilized a modified high-pressure acoustical generator (HPAG, IEStechno, Morgantown, WV) in which bulk CB material (Printex 90®, provided as a gift from Evonik, Frankfurt, Germany) generated ultrafine CB aerosols. The output from the HPAG was fed into a venturi pump (JS-60 M, Vaccon, Medway MA) to further deagglomerate particles. The real-time mass concentration (mg/m³) of aerosolized particles was monitored with a light scattering aerosol monitor (DataRAM, pDr-1500, Thermo Environmental Instruments Inc, Franklin, MA). O₃ was produced by passing HEPA (High-efficiency particulate air) filtered dried air/pure oxygen through a corona discharge type O₃ generator (HTU500AC, Ozone Solutions, Hull, IA). During co-exposures, the O₃ was then mixed with the CB aerosol before entering the exposure chamber. O₃ concentration in the chamber was measured using a calibrated O₃ analyzer (Model 202, 2B Technologies, Inc., Boulder, CO). O₃ monitor calibration was independently verified using Calorimetric ozone gas detector tubes (Sensidyne® LP, St Peterburgh FL). The O₃ levels were maintained by adjusting the flow through the ozone generator based on the real-time readings from the ozone monitor.
Temperature and relative humidity in the exposure chamber were measured (HMT330, Vaisala, Helsinki, Finland) and maintained at 20–22 °C and 50–70% respectively. Exposure chamber and animal housing cages are made with Stainless steel (grade 316) which has excellent compatibility with O₃. The whole-body stainless-steel exposure chamber (Cube 150, IEStechno, Morgantown, WV) individually housed up to 36 mice. Gravimetric measurements of the mass concentration were collected and reported for each exposure and were also used to continually calibrate the DataRAM. Particle size distributions were sampled from the exposure chamber with: 1) an electrical low-pressure impactor (ELPI+, Dakati, Tempera, Finland), 2) an aerosol particle sizer (APS 3321, TSI Inc Shoreview, MN), 3) a scanning mobility particle sizer (SMPS 3938, TSI Inc. Shoreview, MN), and 4) a Nano Micro-orifice Uniform Deposit Impactor (Moudi 115R, MSP Corp, Shoreview, MN). Aerosols were collected on formvar coated copper grids and imaged using JOEL 1400 transmission electron microscope (JOEL, Tokyo, Japan) to characterize morphology. Polycarbonate filters were used to collect the morphology characterization using a field-emission scanning electron microscope (Hitachi S4800, Tokyo, Japan). Elemental composition of particle surfaces was analyzed by X-Ray Photoelectron Spectroscopy (XPS) (Physical Electronics PHI 5000 VersaProbe XPS/UPS). After the aerosol/gas left the chamber it was HEPA and charcoal filtered before entering the house exhaust. Software was developed (IEStechno, Morgantown, WV) to monitor, control and record system parameters during exposures. Various feedback loops were utilized in the software to hold concentration and pressure levels constant during exposures. The pressure in the HPAG was held constant to improve aerosol generation. The chamber pressure was also held constant at zero for animal comfort and to minimize any potential chamber leaks. The DataRAM real-time values were utilized in a feedback loop to hold the aerosol concentration steady by changing the power delivered to the HPAG.
Electron Paramagnetic Resonance (EPR) Spectroscopic Studies: Purified 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) was purchased from Dojindo laboratories, Kumamoto, Japan. Xanthine and xanthine oxidase (XO) from bovine milk (catalog number: X4875) were purchased from Sigma-Aldrich, USA. All the EPR spin trapping and spin probe experiments were carried out in phosphate buffered saline (PBS, pH7.4) pre-treated with Chelex. EPR spin trap experiments were performed using a spin trap DMPO. EPR spectra were recorded using a Bruker EMXnano spectrometer (Bruker BioSciences, Billerica, MA, USA) operating at X-band with a 100 kHz modulation frequency as described previously [30 (link)]. Data acquisition was performed using Bruker Xenon-nano software. Solid/powder samples were loaded directly in to an EPR quartz tube (O.D 4 mm). Liquid samples of 50 μL were loaded into glass capillary tubes that were sealed on one end using Critoseal clay and placed inside the 4 mm (O.D.) EPR quartz tube. The quartz tube was positioned inside the resonator/cavity and EPR spectra were recorded at room temperature. The following settings were used: microwave frequency, 9.615 GHz; sweep width, 100 G (200 G for powder); microwave power, 20 mW; modulation amplitude, 0.5 G (5 G for powder); modulation frequency, 100 kHz; receiver gain, 60 dB; time constant, 41 ms (20.5 ms for powder); conversion time, 15 ms (31 ms for powder), sweep time, 30 s (50 s for powder); number of scans, 1 or 10.
Ferric Reducing Ability of Serum (FRAS) Assay: FRAS assay was performed to study acellular oxidant generation ability of individual and co-exposures by following the already published methodology [31 (link),32 (link)] with a slight modification. In order to accurately mimic the inhalation exposure and eliminate the artifacts that might arise due to interaction with room air, we bubbled aerosols (air, CB, O₃ and CB + O₃) for 5 min through the human serum and proceeded to quantify the changes by exactly following the previously published standardized methodology. In order to validate the occurrence of interactions between O₃ and CB at levels to which population is chronically exposed, we performed FRAS assay using two exposure concentrations for CB (250 μg/m³ (low dose) and 10 mg/m³ (high dose)) and O₃ (200 ppb and 2 ppm).
Animal Exposures and Exposure Conditions: C57BL/6J male mice (8 weeks old) were purchased from Jackson Laboratory (Bar Harbor, ME) and acclimated at the West Virginia University Animal Care Facility before exposure. All animals were maintained in a room with a 12-h light/dark cycle and provided chow and water ad libitum. All the animal procedures were approved by the WVU Animal Care and Use Committee. Animals were exposed for 3 h per day, up to 2 days, to either filtered air, O₃ (2 ppm), CB (10 mg/m³), or CB + O₃ (10 mg/m³ + 2 ppm). EUK-134 (Cayman Chemicals, Ann Arbor, MI) is a catalase/SOD mimetic which prevents oxidative stress. EUK-134 (10 mg/kg) was intraperitoneally injected 30 min prior to exposure and mice were sacrificed 24 h post exposure. TSLP neutralizing antibody (catalog # MAB555-100) and TSLP iso-type antibody (catalog # MAB002) (R&D systems, MN) were administered (0.8 mg/kg) by nasal and oropharyngeal aspiration 1 h before exposure. Antibody administration was performed by both nasal and oropharyngeal routes (half dose by either route) to maximize neutralization of TSLP in both upper and lower airways. TSLP-isotype antibody serves as more specific control for TSLP neutralizing antibody thus eliminating the need of doing PBS only group. The mice were euthanized by intraperitoneal injection of Fatal Plus (250 mg/kg) and analyzed 24 h following the exposure. Details on animal cohorts used for different experiments is presented in Supplemental Table S1. Schematics for exposures are presented in Supplemental Fig. S2.
Carbon Black Lung Burden Quantification: Lung burden was quantified according to a previously described method with slight modifications [33 (link)]. A group of animals (5–7) were euthanized within 15 min of inhalation exposure, lungs were removed, and wet lung weight was quantified. Lung tissue was minced and digested in a 25% KOH/methanol (w/v) solution at 60 °C overnight in a dry heating block. After digestion tubes containing lung samples were vortexed and centrifuged at 16,0000 g for 10 min at 25 °C. Pellet was resuspended in 50% HNO₃/Methanol (v/v) and incubated at 60 °C for 3 h in heating block. Tubes containing lung samples were vortexed and centrifuged at 16,0000 g for 10 min at 25 °C. Pelleted samples and known standards of CB and O₃ interacted CB collected from inhalation chamber were resuspended in surfactant water solution (10% NP-40). Samples and standards (1 mg/mL- 1.56 μg/mL) were spectrophotometrically read at 690 nm, sonicated and re-read till a stable optical density was obtained [33 (link)].
Bronchoalveolar Lavage Fluid (BALF) Collection and Analyses: Whole lung bronchoalveolar lavage (BAL) was performed to collect 3 mL of BALF (3 washes of 1 ml each), pooled and processed for cellular and biochemical analyses as described previously [34 (link),35 (link)]. BALF total cells were quantified using a hemocytometer/automated cell counter (Countess®, Thermofisher Scientific, Waltham, MA). Differential cell counts were performed after cytospin preparation (Cytospin® Thermofisher Scientific, Waltham, MA) as described by us previously [34 (link)]. Cells were stained in Hema3 (Fisher Scientific, Pittsburgh, PA). Percentage of different cells types (macrophages, neutrophils, lymphocytes, eosinophils etc.) were calculated and absolute cell numbers were determined by taking into consideration the volume of lavage fluid collected. Lavage proteins were quantified as a marker for air-blood barrier integrity by Pierce BCA kit (Thermofisher Scientific, Waltham, MA) according to manufacturer's instructions. Lung cell death was estimated by quantifying lactate dehydrogenase (LDH) activity by Cytotox 96 NonRadioactive Cytotoxicity Assay (Promega, Madison, WI) according to manufacturer's instructions and previously published reports [35 (link)].
In Vivo Immunospin Trapping (IST): IST employs antibody-based detection of stable adducts formed by the reaction of free radicals with a spin trap. IST was performed following the methods published by us previously [36 ]. Briefly, mice were intraperitoneally injected with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) 24, 18 and 12 h before exposure (0.5 g/kg for each injection and thus 1.5 g/kg total dose). Mice were exposed by inhalation to filtered air or 10 mg/m³ CB + 2 ppm O₃ co-exposure aerosols for 3 h and sacrificed 24 h post exposure. Lung tissue were immunostained for epithelial cells (EPCAM monoclonal antibody G8.8, Developmental Studies Hybridoma Bank, University of Iowa), actin (Phalloidin, Thermofisher Scientific, Waltham, MA) and nuclei (DAPI, Thermofisher Scientific, Waltham, MA). The rabbit polyclonal anti-DMPO antibody was a kind gift from Dr. Ron Mason (National Institute of Environmental Health Sciences, NIEHS).
Enzyme Linked Immunosorbent Assay (ELISA): ELISA assays were performed for keratinocyte chemoattractant (KC), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-13 (IL-13), interleukin-1β (IL-1β) and thymic stromal lymphopoietin (TSLP) using Duoset sandwich ELISA assay kits (R&D Systems, MN) according to manufacturer's recommendations. Lower limit of detection for these assays were IL-1β (15.6 pg/mL), TNF-α (31.3 pg/mL), KC (15.6 pg/mL), IL-6 (15.6 pg/mL), IL-13 (62.5 pg/mL) and TSLP (15.6 pg/mL).
Lung Histology: Lungs were fixed with 10% neutral buffered formalin instillation through the trachea till fully distended. Hematoxylin and eosin staining was performed on 5 μm thick sections. Tissues were evaluated by a board-certified veterinary pathologist in a blinded fashion.
Real-time PCR Gene Expression: The lung tissues were snap frozen in liquid nitrogen for PCR analyses. Total RNA was extracted using Qiagen RNeasy RNA isolation kit (Qiagen, Germantown, MD) and cDNA was synthesized using Reverse Transcription Kit (High-Capacity cDNA Reverse Transcription Kit, Thermofisher Scientific). Sequences of PCR primers are provided in Supporting information Table S2. PCR reaction was performed in triplicate using AriaMX real time PCR machine (Agilent, Santa Clara CA) using syber green chemistry as described by us previously [37 (link)]. Relative expression level of genes of interest was measured using the comparative threshold method with 18S as internal control. Data were analyzed using ΔΔCt method, where fold change = 2^−ΔΔCt.
Lung Function Measurements: Forced Oscillation technique (FOT) and forced expiration (FE) measurements were performed 24 h post exposure after exposure using FlexiVent mechanical ventilator system (SCIREQ, Inc., Montreal, Canada) equipped with FX1 module as well as negative pressure forced expiration (NPFE) extension. Data was captured and analyzed using flexiWare v7.2 software. Aerosol challenges to (0–100 mg/mL) methacholine (2s each) was performed using synchronized nebulizer activation (Aeroneb Lab nebulizer, 2.5–4 μm; Aerogen, Galway, Ireland) integrated in the inspiratory arm of the Y-tubing. Protocol for these measurements is already described in detail [38 (link)]. Briefly, mice were anesthetized with sodium pentobarbital (70 mg/kg) or urethane (2 mg/kg), a metal tracheal cannula (18 gauge, 0.3 cmH₂O.s/mL resistance) was inserted. Quasi-sinusoidally ventilation with a tidal volume of 10 mL/kg, a frequency of 150 breaths/min, an inspiratory to expiratory ratio of 2:3, and a positive end-expiratory pressure of 3 cmH₂O was performed. After two deep inflations (30 cmH₂O pressure), baseline measurements were performed by applying a broadband forced oscillation waveform inducing frequencies between 0.5 and 19.75 Hz (Prime-8; P8) and were analyzed by the constant-phase model. Newtonian resistance (Rn, airway resistance) was inferred from this data. Overall resistive and elastic properties of the respiratory system were measured using a snapshot 150 perturbation which is a single frequency forced oscillation (matched to subject's ventilation frequency and tidal volume). Data from this Snapshot measurement was fitted to single compartment model and Respiratory system resistance (Rrs) and compliance (Crs) were calculated. Same perturbations were applied in conjunction with increasing doses of methacholine (0–100 mg/mL) to construct dose response [38 (link),39 ]. During these measurements after performing snap shot measurements, a Quick Prime-3 (QP3) perturbation was applied for five runs at approximately 15 s apart, resulting in 5 measurements for each concentration of methacholine. Each sequence was followed by a NPFE measurement taken approximately 15 s after the last FOT measurement using NPFE extension for FlexiVent. Forced Expiratory Volume at 0.1 s (FEV0.1) was measured in triplicate for each dose of methacholine. Moreover, a provocative concentration 20 (PC20), inducing a 20% decrease in FEV0.1 was assessed, by calculating the slope of the dose-response curve of each individual mouse, where the peak responses to MCh were normalized to the FEV0.1 of 0 mg/ml MCh (=100%).
Statistical Analyses: Data are presented as means ± standard deviation (SD) from at least two repeats with a total of 5–10 animals per group. Depending on group size normality of the data was confirmed by suitable normality tests (D'Agostino-Pearson or Shapiro-Wilk). In case of normally distributed data, significant differences between groups were identified by analysis of variance (one-way or two-way, as dictated by experimental design) and Tukey's post hoc test was applied. If data failed normality test, a non-parametric testing was performed, and a Kruskal Wallis post-test for group differences was applied. Individual comparisons between groups were confirmed by Student-t test or Mann-Whitney U test as appropriate. For null hypothesis, a two-tailed p-value of less than 0.05 (95% confidence level) was considered statistically significant. Statistics were performed using GraphPad Prism v7.

Majumder N., Goldsmith W.T., Kodali V.K., Velayutham M., Friend S.A., Khramtsov V.V., Nurkiewicz T.R., Erdely A., Zeidler-Erdely P.C., Castranova V., Harkema J.R., Kelley E.E, & Hussain S. (2021). Oxidant-induced epithelial alarmin pathway mediates lung inflammation and functional decline following ultrafine carbon and ozone inhalation co-exposure. Redox Biology, 46, 102092.

Publication 2021

Most recents protocols related to «Pyrroline»

Quantifying 2-AP in Milled Rice

The 2-acetyl-1-pyrroline (2-AP) in the milled rice was extracted with absolute ethanol and chloroform, and the 2-AP content was determined using a Thermo TSQ 8000 EVO mass spectrometer (Thermo Fisher Scientific Inc., Waltham, MA, USA).

Wei X., Zhang Y., Song X., Zhao L., Zhao Q., Chen T., Lu K., Zhu Z., Huang S, & Wang C. (2024). Silicon and Zinc Fertilizer Application Improves Grain Quality and Aroma in the japonica Rice Variety Nanjing 46. Foods, 13(1), 152.

Publication 2024

Genes Identification in Ulnaria acus Genome

Genes of metacaspases, delta-1-pyrroline-5-carboxylate dehydrogenase, glutathione synthetase, and death-specific protein were searched in the previously published Ulnaria acus (=Synedra acus subsp. radians) genome [71 (link)]. Hmmer software version 3.4. [93 (link)] and hidden Markov models from the Pfam data base [94 (link)] were used for the search.

Bayramova E., Petrova D., Marchenkov A., Morozov A., Galachyants Y., Zakharova Y., Bedoshvili Y, & Likhoshway Y. (2024). Differential Expression of Stress Adaptation Genes in a Diatom Ulnaria acus under Different Culture Conditions. International Journal of Molecular Sciences, 25(4), 2314.

Publication 2024

Synthesis of Bismuth-based Photocatalysts

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Publication 2024

EPR Detection of Hydroxyl Radicals

For EPR detection, 5,5‐Dimethyl‐1‐pyrroline N‐oxide (DMPO) was employed as the •OH trapping agent. Mn‐N/C (1 mg) was added into the NaAc‐HAc buffer (pH 4.0) containing H₂O₂ (1.0 mL) and 100 µm DMPO. The above mixture solutions were transferred to a quartz tube for EPR assay after mixing by sonication for 5 min.

Qiao W., Chen J., Zhou H., Hu C., Dalangood S., Li H., Yang D., Yang Y, & Gui J. (2024). A Single‐Atom Manganese Nanozyme Mn‐N/C Promotes Anti‐Tumor Immune Response via Eliciting Type I Interferon Signaling. Advanced Science, 11(14), 2305979.

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Quantifying Radicals in Humification

Radicals of •OH and SO₄⁻• generated during the humification process were measured, with 5,5-Dimethyl-1-pyrroline-N-oxide (DMPO) as the spin-trapping agent, using an electron paramagnetic resonance spectrometer (EPR spectrometer). To determine the contributions of each radical, quenching tests were conducted by adding 10 mol/L TBA or EtOH to wasted milk before dosing PS and KOH.

Zhu Y., Cao Y., Fu B., Wang C., Shu S., Zhu P., Wang D., Xu H., Zhong N, & Cai D. (2024). Waste milk humification product can be used as a slow release nano-fertilizer. Nature Communications, 15, 128.

Publication 2024

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What is Pyrroline?

What are some common applications of Pyrroline?

What are the different types or variations of Pyrroline?

There are several different types of pyrrolines, including 2-pyrrolline, 3-pyrrolline, and 4-pyrrooline. These variations have slightly different chemical structures and properties, which can influence their reactivity and behavior in different applications. Researchers may need to explore multiple pyrroline variations to find the best fit for their specific needs.

What are some common challenges in working with Pyrroline?

Working with pyrrolines can present some challenges, such as ensuring reproducibility of experimental protocols and identifying the most effective methods for your research goals. Variations in pyrroline structures and properties can also make it difficult to consistently achieve desired outcomes. However, PubCompare.ai's AI-powered platform can help address these challenges by assisting researchers in locating the most effective and reproducible protocols from published literature, pre-prints, and patents.

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More about "Pyrroline"

Pyrrolines are a class of heterocyclic organic compounds containing a five-membered ring with four carbon atoms and one nitrogen atom.
These versatile molecules are found in various biological molecules and compounds, including the amino acid proline.
Pyrrolines exhibit diverse chemical and biological properties, making them important in fields such as medicinal chemistry, biochemistry, and materials science.
Researchers can utilize PubCompare.ai's innovative AI-powered platform to efficiently locate the most effective and reproducible protocols for working with pyrrolines across published literature, pre-prints, and patents.
This can save time and enhance research outcomes.
Related terms and abbreviations: - 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) - 5,5-dimethyl-1-pyrroline N-oxide - 5,5-Dimethyl-1-pyrroline-N-oxide (DMPO) - DMSO (Dimethyl sulfoxide) - FBS (Fetal Bovine Serum) - Milli-Q system (ultrapure water purification system) - EMXplus (electron paramagnetic resonance spectrometer) - Sodium hydroxide (NaOH) Key subtopics: - Pyrroline structure and properties - Biological relevance of pyrrolines - Applications in medicinal chemistry and biochemistry - Utilizing PubCompare.ai for efficient protocol identification - Importance of reproducible and effective research methods With PubCompare.ai's innovative tools, researchers can save time and enhance their outcomes when working with pyrrolines and related compounds.