Macrophage roles in the clearance of apoptotic cells and control of inflammation in the prostate gland after castration.

BACKGROUND: Androgen deprivation results in massive apoptosis in the prostate gland. Macrophages are actively engaged in phagocytosing epithelial cell corpses. However, it is unknown whether microtubule-associated protein 1 light chain 3 alpha (LC3)-associated phagocytosis (LAP) is involved and contribute to prevent inflammation. METHODS: Flow cytometry, RT-PCR and immunohistochemistry were used to characterize the macrophage subpopulation residing in the epithelial layer of the rat ventral prostate (VP) after castration. Stereology was employed to determine variations in the number of ED1 and ED2. Mice were treated with either chloroquine or L-asparagine to block autophagy. RESULTS: M1 (iNOS-positive) and M2 macrophages (MRC1+ and ARG1+) were not found in the epithelium at day 5 after castration. The percentage of CD68+ (ED1) and CD163+ (ED2) phenotypes increased after castration but only CD68+ cells were present in the epithelium. RT-PCR showed increased content of the autophagy markers Bcl1 and LC3 after castration. In addition, immunohistochemistry showed the presence of LC3+ and ATG5+ cells in the epithelium. Double immunohistochemistry showed these cells to be CD68+ /LC3+ , compatible with the LAP phenotype. LC3+ cells accumulate significantly after castration. Chloroquine and L-asparagine administration caused inflammation of the glands at day 5 after castration. CONCLUSIONS: CD68+ macrophages phagocytose apoptotic cell corpses and activate the LAP pathway, thereby contributing to the preservation of a non-inflammed microenvironment. Marked inflammation was detected when autophagy blockers were administered to castrated animals.

Recombinant L-asparaginase 1 from Saccharomyces cerevisiae: an allosteric enzyme with antineoplastic activity.

L-asparaginase (L-ASNase) (EC 3.5.1.1) is an important enzyme for the treatment of acute lymphoblastic leukaemia. Currently, the enzyme is obtained from bacteria, Escherichia coli and Erwinia chrysanthemi. The bacterial enzymes family is subdivided in type I and type II; nevertheless, only type II have been employed in therapeutic proceedings. However, bacterial enzymes are susceptible to induce immune responses, leading to a high incidence of adverse effects compromising the effectiveness of the treatment. Therefore, alternative sources of L-ASNase may be useful to reduce toxicity and enhance efficacy. The yeast Saccharomyces cerevisiae has the ASP1 gene responsible for encoding L-asparaginase 1 (ScASNase1), an enzyme predicted as type II, like bacterial therapeutic isoforms, but it has been poorly studied. Here we characterised ScASNase1 using a recombinant enzyme purified by affinity chromatography. ScASNase1 has specific activity of 196.2 U/mg and allosteric behaviour, like type I enzymes, but with a low K0.5 = 75 µM like therapeutic type II. We showed through site-directed mutagenesis that the T64-Y78-T141-K215 residues are involved in catalysis. Furthermore, ScASNase1 showed cytotoxicity for the MOLT-4 leukemic cell lineage. Our data show that ScASNase1 has characteristics described for the two subfamilies of l-asparaginase, types I and II, and may have promising antineoplastic properties.

Optimization of manganese peroxidase and laccase production in the South American fungus Fomes sclerodermeus (Lév.) Cke.

Fomes sclerodermeus produces manganese peroxidase (MnP) and laccase as part of its ligninolytic system. A Doehlert experimental design was applied in order to find the optimum conditions for MnP and laccase production. The factors studied were Cu(2+), Mn(2+) and asparagine. The present model and data analysis allowed us not only to define optimal media for production of both laccase and MnP, but also to show the combined effects between the factors. MnP was strongly influenced by Mn(2+), which acts as an inducer. Under these conditions Cu(2+) negatively affected MnP activity. At 13 days of growth 0.75 U ml(-1) were produced in the optimized culture medium supplemented with 1 mM MnSO(4) and 4 g l(-1) asparagine. The laccase titer under optimized conditions reached maximum values at 16 days of growth: 13.5 U ml(-1) in the presence of 0.2 mM CuSO(4), 0.4 mM MnSO(4) and 6 g l(-1) asparagine. Mn(2+) promoted production of both enzymes. There were important interactions among the nutrients evaluated, the most significant being those between Cu(2+) and asparagine.

Effect of aspartate and asparagine supplementation on fatigue determinants in intense exercise.

PURPOSE: This study evaluated the effect of aspartate (ASP) and asparagine (ASG) supplementation on fatigue determinants in Wistar rats exercised to exhaustion by swimming. METHODS: The animals were tested for anaerobic threshold (AT) determination and then supplemented with 350 mM ASP + 400 mM ASG x day(-1) (AA group, n = 16) or 2 ml x day(-1) of distillated water (PLC group, n = 16) for 7 days. On the 7th day of supplementation, the animals were divided into 4 new groups and killed at rest (RAA, n = 8; RPLC, n = 8), or immediately after the swimming exercise to exhaustion (EAA, n = 8; EPLC, n = 8). R: No significant differences were observed between amino acids and placebo rest groups for muscle and liver glycogen, blood glucose, lactate, alanine, and glutamine concentrations. However, in the exhaustion groups, the EAA group showed higher exercise time (68.37 +/- 25.42 x 41.12 +/- 13.82 min, p <.05) and lower blood lactate concentration (8.57 +/- 1.92 x 11.28 +/- 2.61 mmol x L(-1), p <.05) than the EPLC group. Moreover, the ASP+ASG supplementation decreased the rate of glycogen degradation of gastrocnemius (1.00 +/- 0.51 x 3.43 +/- 0.99 microg x 100 mg of tissue sample(-1) x min(-1), extensor digitorius longus (5.70 +/- 2.35 x 8.11 +/- 3.97 microg. 100 mg of tissue sample(-1) x min(-1) and liver (0.51 +/- 0.34 x 3.37 +/- 2.31 microg x 100 mg of tissue sample(-1) x min(-1) for EAA. CONCLUSION: These results suggest that ASP+ASG supplementation may increase the contribution of oxidative metabolism in energy production and delay fatigue during exercise performed above the AT.

Possível efeito da suplementaçäo de aminoácidos de cadeia ramificada, aspartato e asparagina sobre o limiar anaeróbio / Possible effect of the supplementation of branched chain aminoacids, aspartate and asparagine on anaerobic threshold

Recentemente o conceito "Limiar Anaeróbio" tem sido muito criticado. As prinipais criticas repousam sobre os mecanismos considerados para o aumento da concentraçäo de lactato sangüíneo, hipóxia muscular principalmente, e sobre a suposta relaçäo de causa-e-efeito entre os limiares metabólico e ventilatório. Apesar de criticado, o conceito Limiar Anaeróbio encontrou muitas aplicaçöes, e por esta razäo, vários estudos foram realizados para facilitar a sua determinaçäo näo invasivamente, a partir de parâmetros ventilatórios e da deflexäo da curva de freqüência cardíaca. Recentemente o uso de aminoácidos tem se difundido largamente entre os praticantes de atividades motoras, tornando-se objeto de estudo para vßrios pesquisadores. Foi proposto que a suplementaçäo de aminoácidos de cadeia ramificada, aspartato e asparagina promove aumento da resistência ao esforço físico prolongado, em decorrência do aumento do conteúdo de glicogênio muscular e síntese de oxaloacetato para manutençäo da atividade do ciclo de Krebs e do próprio metabolismo oxidativo. Com isto o transporte de glicose para o interior da célula muscular diminui, retardando a depleçäo de glicogênio muscular e a acidose metabólica, causas evidentes de fadiga. Em conseqüência, a oxidaçäo de AGLs durante o exercício contínuo moderado aumenta, retardando o acúmulo de lactato sangüíneo e muscular. Isto retardaria o estímulo metabólico para o processo de tamponamento pelo HCO3 e a conseqüente compensaçäo ventilatória para CO2, podendo dissociar os limiares metabólico e ventilatório. A dissociaçäo dos limiares reforçaria as críticas sobre o conceito Limiar Anaeróbio e poderia, inclusive, prejudicar a sua identificaçäo através de outros métodos, como por exemplo, a deflexäo da curva de freqüência cardíaca.

Effect of aspartate, asparagine, and carnitine supplementation in the diet on metabolism of skeletal muscle during a moderate exercise.

The present study examined the effect of diet supplementation of oxaloacetate precursors (aspartate and asparagine) and carnitine on muscle metabolism and exercise endurance. The results suggest that the diet supplementation increased the capacity of the muscle to utilize FFA and spare glycogen. Time to exhaustion was about 40% longer in the experimental group compared to the control, which received commercial diet only. These findings suggest that oxaloacetate may be important to determine the time to exhaustion during a prolonged and moderate exercise.