Abstract In the cells, the last step of the beta-oxidation cycle, aiming at the degradation of fatty acids, is catalyzed by the enzyme named thiolase. It shortens the acyl chain of the acyl-CoA by two carbons. The reaction is reversible, it can proceed for both directions. Thiolases are divided into two categories, synthetic and degradative ones. These two classes of thiolases differ not only by their biological function, but also by their substrate specificity. Degradative thiolases accept substrates with various lengths but synthetic thiolases only accept short chain-acyl-CoAs as a substrate. In humans, at least six isozymes of thiolases are found. The mitochondrial biosynthetic thiolase, T2, differs from other thiolases by getting activated by potassium. In addition, it accepts branched acyl-CoA, namely 2-methyl-acetoacetyl-CoA, as a substrate. This molecule is an important reaction intermediate in the degradation of the amino acid isoleucine. Many human patients have been diagnosed to have a mutation in the gene of T2, and they are treated with a special diet. The results of this theses show that potassium ion rigidifies the groups of the T2 protein involved in the substrate binding. The presence of potassium increases the reaction rate and it also raises the affinity towards some of the substrates. The enzyme mechanistic studies with bacterial thiolase revealed that the oxyanion hole 1, formed by a water molecule and histidine side chain, is important for the synthetic reaction, not so much for the degradative direction. Binding studies showed that both the terminal sulfur of the substrate and the sulfur of the catalytic cysteine are important for the right positioning of the substrate. The electrostatics of the active site also have a significant role in the catalysis. These studies give a good basis for future studies aiming at drug development against this enzyme in pathogenic species.

Ischemic strokes cause excessive release of glutamate, leading to overactivation of N-methyl-D-aspartate receptors (NMDARs) and excitotoxicity-induced neuronal death. For this reason, inhibition of NMDARs has been a central focus in identifying mechanisms to avert this extensive neuronal damage. N-acetyl-aspartyl-glutamate (NAAG), the most abundant neuropeptide in the brain, is neuroprotective in ischemic conditions in vivo. Despite this evidence, the exact mechanism underlying its neuroprotection, and more specifically its effect on NMDARs, is currently unknown due to conflicting results in the literature. Here, we uncover a pH-dependent and subunit specific action of NAAG on NMDARs. Using whole-cell electrophysiological recordings on acute hippocampal slices from adult mice and on HEK293 cells, we found that NAAG increases synaptic GluN2A-containing NMDAR excitatory postsynaptic currents (EPSCs), while effectively decreasing extrasynaptic GluN2B-containing NMDAR EPSCs in physiological pH. Intriguingly, the results of our study further show that in low pH, which is a physiological occurrence during ischemia, NAAG depresses GluN2A-containing NMDAR EPSCs and amplifies its inhibitory effect on GluN2B-containing NMDAR EPSCs, as well as upregulates the surface expression of the GluN2A subunit. Altogether, our data demonstrate that NAAG has differential effects on NMDAR function based on subunit composition and extracellular pH levels. These findings suggest that the role of NAAG as a neuroprotective agent during an ischemic stroke is likely mediated by its ability to reduce NMDAR excitation. The inhibitory effect of NAAG on NMDARs and its enhanced function in acidic conditions makes NAAG a prime therapeutic agent for the treatment of ischemic events.

Some plasticizers are endocrine-disrupting chemicals that cause reproductive toxicity in both males and females. Several chemicals already approved by the Food and Drug Administration have been proposed as substitutes for some of these plasticizers, one example is acetyl tributyl citrate (ATBC). However, no studies have tested whether ATBC causes direct toxicity to the ovary. Ovarian antral follicles are essential for female fertility because they are the major producers of ovarian steroids and are the only follicle type that can ovulate in response to gonadotropin stimulation. Previous studies have used in-vitro ovarian follicle culture as a screening tool to demonstrate that EDCs can cause direct ovarian toxicity. Therefore, we designed this study to test whether exposure to in vitro treatment with ATBC causes ovarian toxicity in CD-1 mice. We mechanically isolated antral follicles from the ovaries of adult CD-1 mice (35-39 days old) and individually exposed them (n=5 cultures with n≥8 follicles per treatment) to supplemented media alone (NT), dimethyl sulfoxide (DMSO, vehicle for ATBC), and ATBC (0.001-100 µg/mL) for 24-72 h. Follicle growth and survival were monitored by measuring follicle diameter and cytotoxicity (compromised membrane integrity; CellTox Green) every 24 h, and assessing number of metabolically active cells (ATP concentration; Promega CellTiterGlo) at the end time point. The DNA synthesis inhibitor hydroxyurea (HU, 100 mM) was used as a positive control for the viability assays. Exposure to ATBC did not affect the ability of antral follicles to increase their diameter over time at all concentrations tested. When stratified by growth pattern, there was not a significant difference in the proportion of follicles growing normally, growing slowly, or not growing following ATBC exposure at all concentrations tested. ATBC treatment did not cause compromised membrane integrity and did not inhibit ATP production at any of the concentrations tested. The positive control, HU, inhibited follicle growth (24-72 h), decreased follicle cell membrane integrity (72 h), and inhibited ATP production (24-72 h). The purpose of this experiment is to evaluate the effects of oral exposure to ATBC in female CD-1 mice. For the in vivo experiments, the female mice (n=22; PND 81) were randomly divided into treatment groups and dosed according to daily body weight with one of the following treatments: corn oil (vehicle, n=7), 5 mg/kg/day ATBC (n=8), or 10 mg/kg/day (n=7) ATBC for 15 consecutive days. Vaginal smears were performed and analyzed daily to measure any change in estrous cyclicity. After the 15th day of dosing the female mice were introduced into an individually housed proven breeder male’s cage. Daily body weight measurements continued and plug checks were performed every morning. Pregnancy and time to conception data did not statistically differ from the vehicle (oil) for all ATBC treatments (days to conception: vehicle 2.43 ± 0.65, 5 mg/kg/day ATBC 2 ± 0.33, 10 mg/kg/day ATBC 2.5 ± 0.22; gestation length: vehicle 19.71 ± 0.18, 5 mg/kg/day ATBC 20 ± 0.19, 10 mg/kg/day ATBC 20 ± 0.00). On the day of parturition the dams and pups were sacrificed; organ weight and gross morphology data was collected for: uterus, kidneys, adrenals, spleen, liver, and ovaries. The data analyzed includes: estrous cyclicity, pre-dosing body weight % gain, dosing body weight % gain, pregnant body weight % gain, organ weight, gestation length, litter size (live vs. dead), litter weight, implantation sites, time to conception, and pup sex ratio. Interestingly, there was an increase in spleen weight at the 5 mg/kg/day treatment when compared to vehicle control-treated spleen weights (spleen weight, Oil: n=7; 5 mg/kg ATBC: n= 8). Treatment with 5 mg/kg/day ATBC caused a significant decrease in average estrous cycle length in days compared to the pre-dosing average estrous cycle length. Animals exposed orally to 10 mg/kg/day ATBC showed a significant decrease in total follicle number (10 mg/kg/day ATBC, 313 ± 20.37) when compared to vehicle (oil) treated mice (vehicle, 433.71 ± 34.85). Also treatment with 10 mg/kg/day ATBC resulted in significant reduction in secondary (101.67 ± 5.7) and late antral (4.17 ± 1.45) follicle numbers when compared to the vehicle-treated mice (secondary follicles: 141.14 ± 14.79, late antral follicles: 7.00 ± 1.54). ATBC treatment with 10 mg/kg/day showed a significantly decreased mean body weight percent gain during pregnancy on days 3, 5, and 14 when compared to animals treated with vehicle (oil), while animals treated with 5 mg/kg/day ATBC showed a significant decrease in weight gained on only day 13 when compared to the vehicle (oil). These novel findings show that ATBC could disrupt ovarian function in mice when exposed to low-dose ATBC.

Para quantificar o teor de lignina em alguns cultivares de aveia, foi utilizado o método espectrofotométrico "lignina solúvel em brometo de acetila – LSBA", onde a lignina é solubilizada em uma solução a 25% de brometo de acetila em ácido acético glacial e em seguida sua absorbância lida a 280 nm; como padrão de referência foi utilizada a lignina extraída da aveia através de uma solução ácida de dioxana. A quantificação da lignina foi realizada em oito cultivares de aveia, nas frações planta inteira, caule e folha, em três estádios de maturidade (45, 55 e 65 dias de idade). Os resultados obtidos foram comparados com três métodos gravimétricos: lignina detergente ácido (LDA), lignina permanganato de potássio (LPer) e lignina Klason (LK). Os quatro métodos estudados mostraram teores de lignina diferentes entre si. Os resultados de LSBA foram superiores para quase todos os cultivares, ocorrendo o inverso para a LDA. Houve uma pequena diferença em relação aos dias de corte, com dados superiores para LSBA, onde o caule apresentou maior variação em relação às demais frações. As curvas de regressão e os espectrogramas indicaram presença de diferenças qualitativas entre as ligninas provenientes da aveia forrageira nos diferentes estádios de maturidade. / To quantify lignin content in some varieties of oat, it was employed the spectrophotometric method acetyl bromide soluble lignin – ABSL where lignin is dissolved into a 25% acetyl bromide solution in glacial acetic acid and its absorbance read at 280 nm; as a standard it was employed a, lignin extracted by mild acidic dioxane solution from the same sample. Lignin quantification was evaluated in eight oat cultivars, divided in three vegetable parts (whole plant, stem and leaf), and three different maturity stages (cuts at 45, 55 and 65 days). Obtained results were compared with three gravimetric methods: acid detergent lignin (ADL), potassium permanganate lignin (PerL) and Klason lignin (KL). All four methodologies yielded different results. Overall, ABSL yielded highest values whereas ADL yielded the lowest ones. There was a slight difference related to maturity, with higher values for ABSL, where stem fraction showed higher variation when comparing to the two other vegetable parts. Regression equations and espectrograms indicated the presence of qualitative differences between lignins from at distinct maturity stages.

No description available.

Acetyl-CoA is a metabolite at the crossroads of central metabolism and the substrate of histone acetyltransferases regulating gene expression. In many tissues fasting or lifespan extending calorie restriction (CR) decreases glucose-derived metabolic flux through ATP-citrate lyase (ACLY) to reduce cytoplasmic acetyl-CoA levels to decrease activity of the p300 histone acetyltransferase (HAT) stimulating pro-longevity autophagy. Because of this, compounds that decrease cytoplasmic acetyl-CoA have been described as CR mimetics. But few authors have highlighted the potential longevity promoting roles of nuclear acetyl-CoA. For example, increasing nuclear acetyl-CoA levels increases histone acetylation and administration of class I histone deacetylase (HDAC) inhibitors increases longevity through increased histone acetylation. Therefore, increased nuclear acetyl-CoA likely plays an important role in promoting longevity. Although cytoplasmic acetyl-CoA synthetase 2 (ACSS2) promotes aging by decreasing autophagy in some peripheral tissues, increased glial AMPK activity or neuronal differentiation can stimulate ACSS2 nuclear translocation and chromatin association. ACSS2 nuclear translocation can result in increased activity of CREB binding protein (CBP), p300/CBP-associated factor (PCAF), and other HATs to increase histone acetylation on the promoter of neuroprotective genes including transcription factor EB (TFEB) target genes resulting in increased lysosomal biogenesis and autophagy. Much of what is known regarding acetyl-CoA metabolism and aging has come from pioneering studies with yeast, fruit flies, and nematodes. These studies have identified evolutionary conserved roles for histone acetylation in promoting longevity. Future studies should focus on the role of nuclear acetyl-CoA and histone acetylation in the control of hypothalamic inflammation, an important driver of organismal aging.

The N-acetyl-L-methionine (NALM) molecule is a methionine (Met) derivative produced via acetylation of the L-Met α-amino group with an N-acetyl group. This molecule has been shown to be bioavailable and capable of fulfilling the dietary requirement for Met in animals and humans. The current experiment was conducted to test a hypothesis that lactating dairy cows fed with NALM would increase milk production by increasing N and energy utilization efficiencies in a dose dependent manner. Eight multiparous Holstein cows that were mid lactation (124 ± 13 days-in-milk) with similar milk production were used in a 4 x 4 Latin square design for 84 d. A developmental NALM product from CJ CheilJedang (Seoul, South Korea) was used as the supplemental source of rumen-protected Met in the present study. Four dietary treatments included 0 g (control), 15 g, 30 g, and 45 g/d/cow of NALM supplementation. Supplementing NALM significantly increased dry matter intake (linear effect; P < 0.01), while milk yield tended to increase quadratically (P = 0.07). A linear decrease in milk fat concentration was seen due to supplementation of NALM in relation to the control ration (P = 0.02). However, milk fat yield was similar across treatments. A trend toward an increase in milk protein yield was observed between the control ration and the ration supplemented with 45 g of NALM (1.18 vs. 1.21 kg/d; P = 0.10). There were no differences in energy-corrected or 3.5% fat-corrected milk yields in response to treatments. It is likely that the supplementation of NALM to mid to late lactating dairy cows may have shifted nutrient and energy utilization toward tissue gain and lactation, which resulted in a decrease in feed efficiency for lactation (P = 0.02). Overall results from the present study suggest that supplementing NALM to mid to late lactating cows can increase milk yield in a dose dependent manner with a shift of net energy partitioning toward milk production and body weight gain. In addition, supplementing NALM increased milk nitrogen (N) output without affecting urinary N excretion.

Acetyl-CoA carboxylase (ACC) catalyzes the formation of malnoyl-CoA, which in turn controls the rate of fatty acid metabolism. ACC beta or 2 has been shown to be localized on the mitochondria in close proximity to carnintine palmitoyl transferase 1 (CPT-1), the enzyme responsible for the influx of acyl-CoA into the matrix where beta oxidation takes place. CPT-1 is inhibited by malonyl-CoA produced by ACC. It has been well documented that AMP activated kinase (AMPK) when activated phosphorylates and inactivates ACC. ACC is controlled allosterically by citrate, which activates, and by palmitoyl-COA, which inhibits. In this study, we asked the question, "Does phosphorylation by AMPK effect the inhibition of ACC by palmitoyl-CoA?" ACC was isolated and then subjected to phosphorylation and activity was measured in varying concentrations of acetyl-CoA and citrate. Phosphoryation reduced the substrate (acetyl-CoA) saturation activity curves for ACC at all levels of palmitoyl-CoA. The Ki for palmitoyl-CoA inhibition of ACC was reduced from 1.7 ± 0.25 µM to 0.85 ± 0.13 uM (p<0.05) as a consequence of phosphorylation. In addition the citrate activation curves for ACC were greatly reduced in the presence of palmitoyl-CoA. The data show that skeletal muscle ACC or ACC-beta is more potently inhibited by palmitoyl-CoA after phosphorylation by AMPK. During long-term exercise when AMPK is activated and muscle palmitoyl-CoA is elevated this may contribute to the low malonyl-CoA and increased fatty acid oxidation.

AMP-activated protein kinase (AMPK), a heterotrimeric protein which serves as a metabolic master switch in skeletal muscle, is a research target for the pharmaceutical treatment and prevention of type 2 diabetes. The expression of all of the isoforms of the subunits of AMPK and AMPK activity are increased in skeletal muscle tissue of hyperthyroid rats. Activity of AMPK is regulated by an upstream kinase (AMPKK). The LKB1-STRAD-MO25 complex is a major AMPKK in skeletal muscle. This experiment was designed to determine whether the increase in AMPK activity is accompanied by a thyroid hormone-induced increase in the expression of the LKB1-STRAD-MO25 complex. LKB1-STRAD-MO25 complex protein expression was determined by Western blots in control rats, in rats given 3 mg of thyroxine and 1 mg of triiodothyronine per kilogram chow for 4 weeks, and in rats given 0.01% propylthiouracil (PTU) in drinking water for 4 weeks. The relative expression of LKB1, MO25, and STRAD, as well as PGC-1α, increased in the soleus of thyroid hormone treated rats vs. the controls. MO25 mRNA increased with thyroid hormone treatment, and STRAD mRNA increased with PTU treatment. Phospho-AMPK and phospho-ACC increased in response to electrical stimulation in muscles of all treatment groups, but was most markedly increased in hyperthyroid rats. Thyroid hormone treatment also increased the amount of phospho-CREB in the soleus, heart, and red quadriceps. These data provide evidence that thyroid hormone partially controls expression of the LKB1-STRAD-MO25 complex, as well the subsequent activation of AMPK.

In the present study, acetyl carnitine, decanoyl carnitine, and palmitoyl carnitine were investigated for their surfactant and solubilizing properties. Decanoyl carnitine and palmitoyl carnitine were found to have surface-active properties while acetyl carnitine was without significant surfactant properties. Surface tension and contact angle were decreased by deanoyl and palmitoyl carnitine. The solubilizing effect of the carnitine analogs on triamcinolone acetonide was investigated. Results suggest that the hydrophobic chain length of carnitine analogs was an important factor affecting their surfactant and solubilizing properties. Palmitoyl and decanoyl carnitine demonstrated a greater solubilizing effect on triamcinolone acetonide than acetyl carnitine.