Incubation Of Homogenates Research Articles

Discovery of Unprecedented Human Stercobilin Conjugates.

Two unique metabolites (M18 and M19) were detected in feces of human volunteers dosed orally with [14C]inavolisib with a molecular ion of parent plus 304 Da. They were generated in vitro by incubation with fecal homogenates and we have evidence that they are formed chemically and possibly enzymatically. Structural elucidation by high resolution mass spectrometry and nuclear magnetic resonance spectroscopy showed that the imidazole ring of inavolisib was covalently bound to partial structures derived from stercobilin, an end-product of heme catabolism produced by the gut microbiome. The structural difference between the two metabolites was the position of methyl and ethyl groups on the pyrrolidin-2-one moieties. We propose a mechanism of M18 and M19 generation from inavolisib and stercobilin whereby nucleophilic attack from the imidazole ring of inavolisib occurs to the bridging carbon of a stercobilin molecule. The proposed mechanism was supported by computational calculations of molecular orbitals and transition geometry. SIGNIFICANCE STATEMENT: We report the characterization of two previously undescribed conjugates of the phosphoinositide 3-kinase inhibitor inavolisib, generated by reaction with stercobilin, an end-product of heme catabolism produced by the gut microbiome. These conjugates were confirmed by generating them using in vitro fecal homogenate incubation via nonenzymatic and possibly enzymatic reactions. Given the unique nature of the conjugate, it is plausible that it may have been overlooked with other small molecule drugs in prior studies.

Effects of Different Na+ Concentrations on cAMP-Dependent Protein Kinase Activity in Postmortem Meat.

cAMP-dependent protein kinase (PKA) activity regulates protein phosphorylation, with Na+ playing a crucial role in PKA activity. The aim of this study was to investigate the effects of different Na+ concentrations on PKA activity and protein phosphorylation level in postmortem muscle. The study consisted of two experiments: (1) NaCl of 0, 20, 100, 200 and 400 mM was added to a muscle homogenate incubation model to analyze the effect of Na+ concentration on PKA activity, and (2) the same concentrations were added to pure PKA in vitro incubation models at 4 °C to verify the effect of Na+ on PKA activity. The PKA activity of the muscle homogenate model increased with storage time in groups with different Na+ concentrations. High concentrations of Na+ inhibited sarcoplasmic protein phosphorylation. The PKA activity at 24 h of storage and the sarcoplasmic protein phosphorylation level at 12 h of storage in the group with 200 mM Na+ was lower than that of the other groups. After 1 h incubation, the PKA activity of samples in the 200 mM Na+ group was inhibited and lower than that in the other Na+ groups in the in vitro incubation model. These results suggest that the Na+ concentration at 200 mM could better inhibit PKA activity. This study provided valuable insights for enhancing curing efficiency and improving meat quality.

Synthesis and tissue distribution of bufadienolides in Bufo bufo gargarizans

This study explored the biosynthesis of bufadienolides(BDs) in Bufo bufo gargarizans to solve the dilemma of the decreasing resources of B. bufo gargarizans and provide a theoretical basis for the sustainable utilization of the resources. Ultra-high performance liquid chromatography-Orbitrap-mass spectrometry(UHPLC-Orbitrap-MS) was employed to detect the synthesis sites of BDs in B. bufo gargarizans, and the results were verified by desorption electrospray ionization-mass spectrometry imaging(DESI-MSI) and homogenate incubation experiments. BDs in B. bufo gargarizans had the highest content in the liver and the highest concentration in the gallbladder, in addition to the parotid gland and skin, which suggested that the liver could synthesize BDs. The results of DESI-MSI also showed that BDs were mainly enriched in the liver rather than the immature parotid gland. The incubation experiment of liver homogenates demonstrated the liver of B. bufo gargarizans had the ability to synthesize BDs. This study showed that the liver was a major organ for the synthesis of BDs in B. bufo gargarizans during metamorphosis, development, and growth, which provided strong theoretical support for the biosynthesis of BDs and the sustainable utilization of B. bufo gargarizans resources.

Discovery of α-Linolenic Acid 16(S)-Lipoxygenase: Cucumber (Cucumis sativus L.) Vegetative Lipoxygenase 3.

The GC-MS profiling of the endogenous oxylipins (Me/TMS) from cucumber (Cucumis sativus L.) leaves, flowers, and fruit peels revealed a remarkable abundance of 16-hydroxy-9,12,14-octadecatrienoic acid (16-HOT). Incubations of homogenates from these organs with α-linolenic acid yielded 16(S)-hydroperoxide (16-HPOT) as a predominant product. Targeted proteomic analyses of these tissues revealed the presence of several highly homologous isoforms of the putative "9S-lipoxygenase type 6". One of these isoenzymes (CsLOX3, an 877 amino acid polypeptide) was prepared by heterologous expression in E. coli and exhibited 16(S)- and 13(S)-lipoxygenase activity toward α-linolenic and linoleic acids, respectively. Furthermore, α-linolenate was a preferred substrate. The molecular structures of 16(S)-HOT and 16(S)-HPOT (Me or Me/TMS) were unequivocally confirmed by the mass spectral data, 1H-NMR, 2D 1H-1H-COSY, TOCSY, HMBC, and HSQC spectra, as well as enantiomeric HPLC analyses. Thus, the vegetative CsLOX3, biosynthesizing 16(S)-HPOT, is the first 16(S)-LOX and ω3-LOX ever discovered. Eicosapentaenoic and hexadecatrienoic acids were also specifically transformed to the corresponding ω3(S)-hydroperoxides by CsLOX3.

Reductive Conversion Leads to Detoxification of Salicortin-like Chemical Defenses (Salicortinoids) in Lepidopteran Specialist Herbivores (Notodontidae).

Lepidopteran specialist herbivores of the Notodontidae family have adapted to thrive on poplar and willow species (Salicaceae). Previous research showed that Cerura vinula, a member of the Notodontidae family occurring throughout Europe and Asia, uses a unique mechanism to transform salicortinoids, the host plant's defense compounds, into quinic acid-salicylate conjugates. However, how the production of this conjugates relates to the detoxification of salicortinoids and how this transformation proceeds mechanistically have remained unknown. To find the mechanisms, we conducted gut homogenate incubation experiments with C. vinula and re-examined its metabolism by analyzing the constituents of its frass. To estimate the contribution of spontaneous degradation, we examined the chemical stability of salicortinoids and found that salicortinoids were degraded very quickly by midgut homogenates and that spontaneous degradation plays only a marginal role in the metabolism. We learned how salicortinoids are transformed into salicylate after we discovered reductively transformed derivatives, which were revealed to play key roles in the metabolism. Unless they have undergone the process of reduction, salicortinoids produce toxic catechol. We also studied constituents in the frass of the Notodontidae species Cerura erminea, Clostera anachoreta, Furcula furcula, Notodonta ziczac, and Pheosia tremula, and found the same metabolites as those described for C. vinula. We conclude that the process whereby salicortinoids are reductively transformed represents an important adaption of the Notodontidae to their Salicaceae host species.

Sorafenib versus Lenvatinib Causes Stronger Oxidative Damage to Membrane Lipids in Noncancerous Tissues of the Thyroid, Liver, and Kidney: Effective Protection by Melatonin and Indole-3-Propionic Acid.

Sorafenib and lenvatinib are multi-targeted tyrosine kinase inhibitors which are currently approved to treat advanced hepatocellular carcinoma, renal cell carcinoma and radioiodine-refractory differentiated thyroid carcinoma. However this treatment is often limited due to common adverse events which may occur via oxidative stress. The study aims to compare sorafenib- and lenvatinib-induced oxidative damage to membrane lipids (lipid peroxidation, LPO) in homogenates of porcine noncancerous tissues of the thyroid, the liver, and the kidney and to check if it can be prevented by antioxidants melatonin and indole-3-propionic acid (IPA). Homogenates of individual tissues were incubated in the presence of sorafenib or lenvatinib (1 mM, 100 µM, 10 µM, 1 µM, 100 nM, 10 nM, 1 nM, 100 pM) together with/without melatonin (5.0 mM) or IPA (5.0 mM). The concentration of malondialdehyde + 4-hydroxyalkenals, as the LPO index, was measured spectrophotometrically. The incubation of tissue homogenates with sorafenib resulted in a concentration-dependent increase in LPO (statistically significant for concentrations of 1mM and 100 µM in the thyroid and the liver, and of 1 mM, 100 µM, and 10 µM in the kidney). The incubation of thyroid homogenates with lenvatinib did not change LPO level. In case of the liver and the kidney, lenvatinib increased LPO but only in its highest concentration of 1 mM. Melatonin and IPA reduced completely (to the level of control) sorafenib- and lenvatinib-induced LPO in all examined tissues regardless of the drug concentration. In conclusion, sorafenib comparing to lenvatinib is a stronger damaging agent of membrane lipids in noncancerous tissues of the thyroid, the liver, and the kidney. The antioxidants melatonin and IPA can be considered to be used in co-treatment with sorafenib and lenvatinib to prevent their undesirable toxicity occurring via oxidative stress.

A Pilot Study towards the Impact of Type 2 Diabetes on the Expression and Activities of Drug Metabolizing Enzymes and Transporters in Human Duodenum.

To characterize effects of type 2 diabetes (T2D) on mRNA expression levels for 10 Cytochromes P450 (CYP450s), two carboxylesterases, and three drug transporters (ABCB1, ABCG2, SLCO2B1) in human duodenal biopsies. To compare drug metabolizing enzyme activities of four CYP450 isoenzymes in duodenal biopsies from patients with or without T2D. mRNA levels were quantified (RT-qPCR) in human duodenal biopsies obtained from patients with (n = 20) or without (n = 16) T2D undergoing a scheduled gastro-intestinal endoscopy. CYP450 activities were determined following incubation of biopsy homogenates with probe substrates for CYP2B6 (bupropion), CYP2C9 (tolbutamide), CYP2J2 (ebastine), and CYP3A4/5 (midazolam). Covariables related to inflammation, T2D, demographic, and genetics were investigated. T2D had no major effects on mRNA levels of all enzymes and transporters assessed. Formation rates of metabolites (pmoles mg protein−1 min−1) determined by LC-MS/MS for CYP2C9 (0.48 ± 0.26 vs. 0.41 ± 0.12), CYP2J2 (2.16 ± 1.70 vs. 1.69 ± 0.93), and CYP3A (5.25 ± 3.72 vs. 5.02 ± 4.76) were not different between biopsies obtained from individuals with or without T2D (p > 0.05). No CYP2B6 specific activity was measured. TNF-α levels were higher in T2D patients but did not correlate with any changes in mRNA expression levels for drug metabolizing enzymes or transporters in the duodenum. T2D did not modulate expression or activity of tested drug metabolizing enzymes and transporters in the human duodenum. Previously reported changes in drug oral clearances in patients with T2D could be due to a tissue-specific disease modulation occurring in the liver and/or in other parts of the intestines.

Elucidation of the biosynthetic pathways of boldenone in the equine testis

Boldenone is an anabolic-androgenic steroid that is prohibited in equine sports. Urine from the uncastrated male horse contains boldenone that is thought to be of endogenous origin and thus a threshold (‘cut-off’) concentration has been adopted internationally for free and conjugated boldenone to help distinguish cases of doping from its natural production. The testis is likely to be a source of boldenone. Qualitative analysis was performed on extracts of equine testicular homogenates (n = 3 horses) incubated non-spiked and in the presence of its potential precursors using liquid chromatography tandem mass spectrometry (LC–MS/MS) and LC high resolution mass spectrometry (LC–HRMS). Samples were analysed both underivatised and derivatised to increase the certainty of identification. In addition to previously reported endogenous steroids, analysis of non-spiked testicular tissue samples demonstrated the presence of boldenone and boldienone at trace levels in the equine testis. Incubation of homogenates with deuterium or carbon isotope labelled testosterone and androstenedione resulted in the matching stable isotope analogues of boldenone and boldienone being formed. Additionally, deuterium and carbon labelled 2-hydroxyandrostenedione was detected, raising the possibility that this steroid is a biosynthetic intermediate. In conclusion, boldenone and boldienone are naturally present in the equine testis, with the biosynthesis of these steroids arising from the conversion of testosterone and androstenedione. However, additional work employing larger numbers of animals, further enzyme kinetic experiments and pure reference standards for 2-OH androstenedione isomers would be required to better characterize the pathways involved in these transformations.

Vasodilation Elicited by Isoxsuprine, Identified by High-Throughput Virtual Screening of Compound Libraries, Involves Activation of the NO/cGMP and H₂S/KATP Pathways and Blockade of α₁-Adrenoceptors and Calcium Channels.

Recently, our research group demonstrated that uvaol and ursolic acid increase NO and H2S production in aortic tissue. Molecular docking studies showed that both compounds bind with high affinity to endothelial NO synthase (eNOS) and cystathionine gamma-lyase (CSE). The aim of this study was to identify hits with high binding affinity for the triterpene binding-allosteric sites of eNOS and CSE and to evaluate their vasodilator effect. Additionally, the mechanism of action of the most potent compound was explored. A high-throughput virtual screening (HTVS) of 107,373 compounds, obtained from four ZINC database libraries, was performed employing the crystallographic structures of eNOS and CSE. Among the nine top-scoring ligands, isoxsuprine showed the most potent vasodilator effect. Pharmacological evaluation, employing the rat aorta model, indicated that the vasodilation produced by this compound involved activation of the NO/cGMP and H2S/KATP signaling pathways and blockade of α1-adrenoceptors and L-type voltage-dependent Ca2+ channels. Incubation of aorta homogenates in the presence of isoxsuprine caused 2-fold greater levels of H2S, which supported our preliminary in silico data. This study provides evidence to propose that the vasodilator effect of isoxsuprine involves various mechanisms, which highlights its potential to treat a wide variety of cardiovascular diseases.

Involvement of RhoA/Rho-kinase in l-cysteine/H2S pathway-induced inhibition of agonist-mediated corpus cavernosal smooth muscle contraction

Rho-kinase activity is a key regulator in the maintenance of corporal vasoconstriction and penile detumescense. Also, importance of l-cysteine/H2S pathway in erectile tissue has been shown; however it is currently unknown the role RhoA/Rho-kinase pathway in H2S-induced inhibition in cavernosal tissue. We investigated the role of RhoA/Rho-kinase pathway in the inhibitory effect of l-cysteine and NaHS, as endogenous and exogenous H2S, respectively, on phenylephrine-induced contractions of mouse cavernosal strips. Phenylephrine, α1 receptor agonist, (10 nM-100 μM) induced a concentration-dependent contraction in CC. l-cysteine (endogenous H2S substrate; 10 mM) and exogenous H2S (NaHS; 1 mM) significantly inhibited the contractile response to phenylephrine (P < 0.05). Inhibition of CSE and CBS enzymes by PAG (10 mM) and AOAA (1 mM), respectively, significantly reversed the inhibitory effects of l-cysteine on phenylephrine-induced contraction (P < 0.05). Y-27632 (1 μM), a specific Rho-kinase inhibitor, significantly augmented the inhibitory effect of l-cysteine and NaHS on phenylephrine-induced contraction, and this inhibition was reversed by PAG and AOAA (P < 0.05). In addition, the formation of H2S was increased by approximately 1.8 fold over basal values after incubation of tissue homogenates with l-cysteine. Y-27632 significantly increased both basal and l-cysteine-induced H2S formation and this augmentation diminished by PAG and AOAA (P < 0.05). Furthermore, the pMYPT-1 expression was significantly decreased by l-cysteine, NaHS or Y-27632 alone. Also, pMYPT-1 expression was completely abolished by the l-cysteine/NaHS plus Y-27632 combination, and this inhibition was reversed by PAG and AOAA (P < 0.05). These results suggest that there is an interaction between Rho-kinase and H2S pathways. Rho-kinase may be, at least in part, inhibits CSE/CBS enzymes in mouse corpus cavernosal tissue; however, it is not excluded the other kinases such as PKC and Zip-kinase.

Detection of Divinyl Ether Synthase Activity and a Novel Oxylipin (1'Z)–Colneleic Acid in Asparagus (Asparagus officinalis L.)

The activity of divinyl ether synthase (EC: 4.2.1.121), an enzyme involved in the lipoxygenase cascade of unsaturated fatty acids in plants, has been found in the roots and stalks of asparagus Asparagus officinalis L. Incubation of homogenates of roots and/or etiolated stalks of Asparagus officinalis L. with linoleic acid results in the formation of mainly divinyl ether (9Z,11Z,1'E)-12-(1'-hexenyloxy)-9,11-dodecadienoic acid ((11Z)-etheroleic acid), as well as a minor amount of its geometric isomer (9Z,11E,1'E)-12-(1'-hexenyloxy)-9,11-dodecadienoic acid (etheroleic acid). These compounds are derived from linoleic acid by the sequential action of regiospecific 13-lipoxygenase and divinyl ether synthase. Moreover, a minor compound, whose amount increases after the incubation of homogenates with the 9-hydroperoxide of linoleic acid ((9S,10E,12Z)-9-hydroperoxy(10,12)–octadecadienoic acid), has been found. This compound has been identified as (8E,1'Z,3'Z)-9-(1',3'-nonadienyloxy)-8-nonenoic acid ((1'Z)-colneleic acid). These data suggest that nonphotosynthetic tissues of asparagus exhibit the activity of a novel 13/9-specific divinyl ether synthase.

Uptake, elimination and biotransformation of N-ethyl perfluorooctane sulfonamide (N-EtFOSA) by the earthworms (Eisenia fetida) after in vivo and in vitro exposure

N-ethyl perfluorooctane sulfonamide (N-EtFOSA) is commonly known as the active ingredient of sulfluramid. It can be degraded to perfluorooctane sulfonic acid (PFOS) in biota and environment. Earthworms (Eisenia fetida) were exposed with N-EtFOSA to examine the bioaccumulation, elimination and metabolism of N-EtFOSA by the earthworms after in vivo and in vitro exposure. N-EtFOSA could be biodegraded in quartz sands to perfluorooctane sulfonamide (FOSA) and PFOS. In the in vivo tests, in addition to parent N-EtFOSA, three metabolites, including perfluorooctane sulfonamide acetate (FOSAA), FOSA and PFOS also accumulated in earthworms as a result of N-EtFOSA biotransformation, with FOSA as the predominant metabolite. The bioaccumulation factor (BAF) and uptake rate coefficient (ku) of N-EtFOSA from sand were 20.4 and 2.41·d−1, respectively. The elimination rate constants (ke) decreased in the order FOSAA (0.130·d−1) > N-EtFOSA (0.118·d−1) > FOSA (0.073·d−1) > PFOS (0.051·d−1). The biotransformation of N-EtFOSA in earthworm was further confirmed by the in vitro test involving incubation of earthworm homogenates with N-EtFOSA. This work provides evidence on the accumulation and transformation of N-EtFOSA in terrestrial invertebrates and will be helpful to explore the indirect sources of FOSA and PFOS in environmental biota.

Cytosolic β-glucosidase inhibition and renal blood flow suppression are leading causes for the enhanced systemic exposure of salidroside in hypoxic rats.

The promising benefits of salidroside (SAL) in alleviating high altitude sickness boost investigations on its pharmacokinetics and biological activity. However, the transportation and disposition process of SAL under hypoxic conditions has never been explored. The current study was proposed to investigate the pharmacokinetics of SAL in hypoxic rats and to explore the underlying mechanisms for the distinct metabolic fate of SAL under hypoxia. Pharmacokinetic studies on SAL was conducted in both hypoxic and normoxic rats. The transport properties of SAL were investigated on both hypoxic and normoxic Caco-2 monolayer models. Enzymes involved in SAL metabolism were identified and the effects of hypoxia on these enzymes were assessed by real-time PCR, western blotting analyses, and rat liver homogenate incubation. The renal clearance (CLr) of SAL, effective renal plasma flow (ERPF) and glomerular filtration rate (GFR) in both hypoxic and normoxic rats were also determined for renal function assessment. It was found that the systemic exposure of SAL in hypoxic rats was remarkably higher than that in normoxic rats. The barrier function of Caco-2 monolayer was weakened under hypoxia due to the impaired brush border microvilli and decreased expression of tight junction protein. Hepatic metabolism of SAL in hypoxic rats was attenuated due to the reduced activity of cytosolic β-glucosidase (CBG). Moreover, CLr of SAL was reduced in hypoxic rats due to the suppressed ERPF. Our findings suggest the potential need for dose-adjustment of SAL or its structural analogs under hypoxic conditions.

4-Methylzymosterone and Other Intermediates of Sterol Biosynthesis from Yeast Mutants Engineered in the ERG27 Gene Encoding 3-Ketosteroid Reductase.

Studies in the post-squalene section of sterol biosynthesis may be hampered by the poor availability of authentic standards. The present study used different yeast strains engineered in 3-ketosteroid reductase (Erg27p) to obtain radioactive and non-radioactive intermediates of sterol biosynthesis hardly or not available commercially. Non-radioactive 3-keto 4-monomethyl sterones were purified from non-saponifiable lipids extracted from cells bearing point-mutated 3-ketosteroid reductase. Two strategies were adopted to prepare the radioactive compounds: (1) incubation of cell homogenates of an ERG27-deletant strain with radioactive lanosterol, (2) incubation of growing cells of a strain expressing point-mutated 3-ketosteroid reductase with radioactive acetate. Chemical reduction of both radioactive and non-radioactive 3-keto sterones gave the physiological 3-β OH sterols, as well as the non-physiological 3-α OH isomers. This combined biological and chemical preparation procedure provided otherwise unavailable or hardly available 4-mono-methyl intermediates of sterol biosynthesis, paving the way for research into their roles in physiological and pathological conditions.

Resistance exercise training and invitro skeletal muscle oxidative capacity in older adults.

Whether resistance exercise training (RET) improves skeletal muscle substrate oxidative capacity and reduces mitochondrial production of reactive oxygen species in older adults remains unclear. To address this, 19 older males (≥60 years) were randomized to a RET (n = 11) or to a waitlist control group (n = 8) that remained sedentary for 12 weeks. RET was comprised of three upper body and four lower body movements on resistance machines. One set of 8–12 repetitions to failure of each movement was performed on three nonconsecutive days/week. Improvements in chest press and leg press strength were assessed using a three‐repetition maximum (3 RM). Body composition was assessed via dual energy X‐ray absorptiometry. Muscle biopsies were obtained from the vastus lateralis muscle at baseline and at both 3 weeks and 12 weeks. Palmitate and pyruvate oxidation rates were measured from the 14 CO 2 produced from [1‐14C] palmitic acid and [U‐14C] pyruvate, respectively, during incubation of muscle homogenates. PGC‐1α, TFAM, and PPAR δ levels were quantified using qRT‐PCR. Citrate synthase (CS) and β‐HAD activities were determined spectrophotometrically. Mitochondrial production of reactive oxygen species (ROS) were assessed using the Amplex Red Hydrogen Peroxide/Peroxidase assay. There were no significant changes in body weight or body composition following the intervention. Chest press and leg press strength (3RM) increased ~34% (both P < 0.01) with RET. There were no significant changes in pyruvate or fatty acid oxidation or in the expression of target genes with the intervention. There was a modest increase (P < 0.05) in β HAD activity with RET at 12 weeks but the change in CS enzyme activity was not significant. In addition, there were no significant changes in ROS production in either group following RET. Taken together, the findings of this study suggest that 12 weeks of low volume RET does not increase skeletal muscle oxidative capacity or reduce ROS production in older adults.

Inhibitory effect of aqueous extract of different parts of Gossypium herbaceum on key enzymes linked with type 2 diabetes and oxidative stress in rat pancreas in vitro

This study sought to determine the inhibitory effect of aqueous extract of different parts (bark, leaf, and flower) of cotton plant (Gossypium herbaceum) on key enzymes linked with type 2 diabetes and oxidative stress in rat pancreas in vitro. The aqueous extract (1:10 w/v) of Gossypium herbaceum was prepared and the ability of the extract to inhibit the activity of α-amylase and α-glucosidase as well as activities of pro-oxidant Fe2+-induced lipid peroxidation was determined spectrophotometrically. The results revealed that the three varieties were able to inhibit the activity of α-amylase and α-glucosidase in rat's pancreas in a dose dependent manner (0–88.8 mg/ml). Also, the incubation of pancreas tissue homogenate in the presence of Fe2+ caused a significant increase (233.3%) in the malondialdehyde (MDA) content of pancreas homogenate, nevertheless, the introduction of the aqueous extract inhibited MDA production dose dependently (0–33.33 mg/ml) and also exhibited further antioxidant properties as represented by their high radical scavenging and Fe2+ chelating abilities. Inhibition of α-amylase and α-glucosidase activities has been the primary treatment for the management/prevention of type 2 diabetes. Therefore, the α-amylase and α-glucosidase inhibitory activities of aqueous extracts of different parts of Gossypium herbaceum in rat pancreas and prevention of lipid peroxidation in the tissue may be attributed to the presence of polyphenol content of the plant.

Preovulatory Maternal Effects on Intra- and Interpopulation Variation in Sex Ratios and Phenotypic Characteristics of Magdalena River Turtles (Podocnemis lewyana)

Abstract: In oviparous species, preovulatory maternal effects such as investment in yolk quantity and composition or levels of yolk steroid hormones might potentially affect hatchling body sizes, growth rates, performance, and sex in species with temperature-dependent sex determination. We investigated the effects of egg mass and levels of testosterone and 17β-estradiol in the yolk on the phenotypes of hatchling Magdalena River Turtles (Podocnemis lewyana) in three populations in the Magdalena River drainage of northern Colombia. We hypothesized that, under homogeneous incubation conditions, the differences documented among clutches would be attributable to familial effects, caused by either heritable genetic factors or preovulatory maternal effects. Eggs were incubated artificially at the pivotal temperature for the species (33.4°C) and hatchlings were reared in the laboratory for 2 mo. Two eggs from each clutch were tested for testosterone and 17β-estradiol levels. Sex ratios, hatchling size, mass, and...

Contrasting effects of Na+, K+-ATPase activation on seizure activity in acute versus chronic models

Epilepsy is a life-shortening brain disorder affecting approximately 1% of the worldwide population. Most epilepsy patients are refractory to currently available antiepileptic drugs (AEDs). Knowledge about the mechanisms underlying seizure activity and probing for new AEDs is fundamental to the discovery of new therapeutic strategies. Brain Na+, K+-ATPase activity contributes to the maintenance of the electrochemical gradients underlying neuronal resting and action potentials as well as the uptake and release of neurotransmitters. Accordingly, a decrease of Na+, K+-ATPase increases neuronal excitability and may predispose to appearing of seizure activity. In the present study, we tested the hypothesis that activation of Na+, K+-ATPase activity with a specific antibody (DRRSAb) raised against a regulatory site in the α subunit would decrease seizure susceptibility. We found that incubation of hippocampal homogenates with DRRSAb (1μM) increased total and α1 Na+, K+-ATPase activities. A higher concentration (3μM) increased total, α1 and α2/α3 Na+, K+-ATPase activities. Intrahippocampal injection of DRRSAb decreased the susceptibility of post status epilepticus animals to pentylenetetrazol (PTZ)-induced myoclonic seizures. In contrast, administration of DRRSAb into the hippocampus of naïve animals facilitated the appearance of PTZ-induced seizures. Quantitative analysis of hippocampal electroencephalography (EEG) recordings revealed that DRRSAb increased the percentage of total power contributed by the delta frequency band (0–3Hz) to a large irregular amplitude pattern of hippocampal EEG. On the other hand, we found no DRRSAb-induced changes regarding the theta functional state. Further studies are necessary to define the potential of Na+, K+-ATPase activation as a new therapeutic approach for seizure disorders.

The effects of endogenous non-peptide molecule isatin and hydrogen peroxide on proteomic profiling of rat brain amyloid-β binding proteins: relevance to Alzheimer's disease?

The amyloid-β peptide is considered as a key player in the development and progression of Alzheimer’s disease (AD). Although good evidence exists that amyloid-β accumulates inside cells, intracellular brain amyloid-binding proteins remain poorly characterized. Proteomic profiling of rat brain homogenates, performed in this study, resulted in identification of 89 individual intracellular amyloid-binding proteins, and approximately 25% of them were proteins that we had previously identified as specifically binding to isatin, an endogenous neuroprotector molecule. A significant proportion of the amyloid-binding proteins (more than 30%) are differentially expressed or altered/oxidatively modified in AD patients. Incubation of brain homogenates with 70 µM hydrogen peroxide significantly influenced the profile of amyloid-β binding proteins and 0.1 mM isatin decreased the number of identified amyloid-β binding proteins both in control and hydrogen peroxide treated brain homogenates. The effects of hydrogen peroxide and isatin have been confirmed in optical biosensor experiments with purified glyceraldehyde-3-phosphate dehydrogenase, one of the known crucial amyloid-β binding proteins (also identified in this study). Data obtained suggest that isatin protects crucial intracellular protein targets against amyloid binding, and possibly favors intracellular degradation of this protein via preventing formation of amyloid-β oligomers described in the literature for some isatin derivatives.

Methylated Medium‐ and Long‐Chain Fatty Acids Provide Novel Sources of Anaplerotic Carbon for Fasting or Exercising Mice (LB438)

We hypothesized that methylated fatty acids (e.g. 2‐methylpentanoic acid (2MeP), phytanic or pristanic acids) would provide anaplerotic carbon to the tricarboxylic acid (TCA) cycle and thereby enhance fatty acid oxidation, especially under conditions when TCA cycle intermediates are depleted. The optimal dose of 2MeP and pristanic acid for increasing in vitro [1‐14C]‐oleic acid oxidation in liver or skeletal muscle homogenates from fasted mice (n=4) was assessed using doses of 0, 0.25, 0.5, 0.75, or 1.0 mM. The minimum dose (0.25 mM) maximally stimulated liver tissue oxidation of [1‐14C]‐oleate to 14CO2 by 2‐fold (P &lt; 0.05), but no stimulation was detected in muscle. Similar incubations with 0.25 mM 2MeP, hexanoate, palmitate, phytanic acid, or pristanic acid or 0.1 mM malate or propionyl‐CoA were conducted with liver and skeletal muscle homogenates from acutely exercised or sedentary mice. Regardless of exercise, incubation of liver homogenates with 2MeP increased [1‐14C]‐oleate oxidation to 14CO2 by 60% over control incubations containing hexanoate (P &lt; 0.05). Phytanic acid treatment increased [14C]‐acid‐soluble product accumulation in liver tissue as compared to palmitate (P &lt; 0.05). Anaplerotic stimulation was unaffected by exercised state (P &gt; 0.05) and not evidenced in muscle homogenates. Results were consistent with our hypothesis that methyl‐branched fatty acids may provide anaplerotic carbon and thereby stimulate in vitro fatty acid oxidation.Grant Funding Source: North Carolina Agricultural Research Service and by USDA‐AFRI training grant # 2010‐65200‐20354