Expression of fatty acid-CoA ligase 4 during development and in brain

Fatty acid utilization is initiated by fatty acid-CoA ligase, which converts free fatty acids into fatty acyl-CoA esters. We have cloned previously the human long-chain fatty acid-CoA ligase 4 (FACL4), which is a central enzyme in controlling the free arachidonic acid level in cells and thereby regulating eicosanoid production. We report here the expression of this gene in tissues, particularly in different parts of the brain. We found that FACL4 encoded a 75 kDa enzyme and that there was a modified translation product expressed in the brain. FACL4 was expressed in early stages of development with a significant amount of FACL4 mRNA detected in an E7 mouse embryo. In addition, FACL4 was highly expressed in both adult and newborn mouse brain especially in the granule cells of the dentate gyrus and the pyramidal cell layer of CA1 in hippocampus, and the granular cell layer and Purkinje cells of the cerebellum.     

Antiproliferative and apoptotic effects of tocopherols and tocotrienols on preneoplastic and neoplastic mouse mammary epithelial cells

Studies were conducted to determine the comparative effects of tocopherols and tocotrienols on preneoplastic (CL-S1), neoplastic (-SA), and highly malignant (+SA) mouse mammary epithelial cell growth and viability in vitro. Over a 5-day culture period, treatment with 0-120 microM alpha- and gamma-tocopherol had no effect on cell proliferation, whereas growth was inhibited 50% (IC50) as compared with controls by treatment with the following: 13, 7, and 6 microM tocotrienol-rich-fraction of palm oil (TRF); 55, 47, and 23 microM delta-tocopherol; 12, 7, and 5 microM alpha-tocotrienol; 8, 5, and 4 microM gamma-tocotrienol; or 7, 4, and 3 microM delta-tocotrienol in CL-S1, -SA and +SA cells, respectively. Acute 24-hr exposure to 0-250 microM alpha- or gamma-tocopherol (CL-S1, -SA, and +SA) or 0-250 microM delta-tocopherol (CL-S1) had no effect on cell viability, whereas cell viability was reduced 50% (LD50) as compared with controls by treatment with 166 or 125 microM delta-tocopherol in -SA and +SA cells, respectively. Additional LD50 doses were determined as the following: 50, 43, and 38 microM TRF; 27, 28, and 23 microM alpha-tocotrienol; 19, 17, and 14 microM gamma-tocotrienol; or 16, 15, or 12 microM delta-tocotrienol in CL-S1, -SA, and +SA cells, respectively. Treatment-induced cell death resulted from activation of apoptosis, as indicated by DNA fragmentation. Results also showed that CL-S1, -SA, and +SA cells preferentially accumulate tocotrienols as compared with tocopherols, and this may partially explain why tocotrienols display greater biopotency than tocopherols. These data also showed that highly malignant +SA cells were the most sensitive, whereas the preneoplastic CL-S1 cells were the least sensitive to the antiproliferative and apoptotic effects of tocotrienols, and suggest that tocotrienols may have potential health benefits in preventing and/or reducing the risk of breast cancer in women.     

Glossina morsitans morsitansandGlossina palpalis palpalis:Dosage Compensation Raises Questions about the Milligan Model for Control of Trypanosome Development

Gooding, R. H., and McIntyre, G. S. 1998.Glossina morsitans morsitansandGlossina palpalis palpalis: Dosage compensation raises questions about the Milligan model for control of trypanosome development.Experimental Parasitology90, 244–249. Evidence that dosage compensation occurs in tsetse flies was obtained by comparing the activities of X chromosome-linked enzymes, arginine phosphokinase and glucose-6-phosphate dehydrogenase inGlossina m. morsitansand hexokinase and phosphoglucomutase inGlossina p. palpalis, with the activity of an autosome-linked enzyme, malate dehydrogenase, in each species. The shortcomings of the X chromosome model for the control ofTrypanozoonmaturation in tsetse are discussed in light of these findings and previously published reports on the lack of fitness effects of matureTrypanozooninfections in tsetse and on published results on antitrypanosomal factors in male and female tsetse flies.     

Livers with Constitutive mTORC1 Activity Resist Steatosis Independent of Feedback Suppression of Akt

Insulin resistance is an important contributing factor in non-alcoholic fatty liver disease. AKT and mTORC1 are key components of the insulin pathway, and play a role in promoting de novo lipogenesis. However, mTORC1 hyperactivity per se does not induce steatosis in mouse livers, but instead, protects against high-fat diet induced steatosis. Here, we investigate the in vivo mechanism of steatosis-resistance secondary to mTORC1 activation, with emphasis on the role of S6K1-mediated feedback inhibition of AKT. Mice with single or double deletion of Tsc1 and/or S6k1 in a liver-specific or whole-body manner were generated to study glucose and hepatic lipid metabolism between the ages of 6–14 weeks. Following 8 weeks of high-fat diet, the Tsc1-/-;S6k1-/- mice had lower body weights but higher liver TG levels compared to that of the Tsc1-/- mice. However, the loss of S6k1 did not relieve feedback inhibition of Akt activity in the Tsc1-/- livers. To overcome Akt suppression, Pten was deleted in Tsc1-/- livers, and the resultant mice showed improved glucose tolerance compared with the Tsc1-/- mice. However, liver TG levels were significantly reduced in the Tsc1-/-;Pten-/- mice compared to the Pten-/- mice, which was restored with rapamycin. We found no correlation between liver TG and serum NEFA levels. Expression of lipogenic genes (Srebp1c, Fasn) were elevated in the Tsc1-/-;Pten-/- livers, but this was counter-balanced by an up-regulation of Cpt1a involved in fatty acid oxidation and the anti-oxidant protein, Nrf2. In summary, our in vivo models showed that mTORC1-induced resistance to steatosis was dependent on S6K1 activity, but not secondary to AKT suppression. These findings confirm that AKT and mTORC1 have opposing effects on hepatic lipid metabolism in vivo.     

Implementation of an Electronic Monitoring and Evaluation System for the Antiretroviral Treatment Programme in the Cape Winelands District,South Africa: A Qualitative Evaluation

BackgroundA pragmatic three-tiered approach to monitor the world’s largest antiretroviral treatment (ART) programme was adopted by the South African National Department of Health in 2010. With the rapid expansion of the programme, the limitations of the paper-based register (tier 1) were the catalyst for implementation of the stand-alone electronic register (tier 2), which offers simple digitisation of the paper-based register. This article engages with theory on implementation to identify and contextualise enabling and constraining factors for implementation of the electronic register, to describe experiences and use of the register, and to make recommendations for implementation in similar settings where standardisation of ART monitoring and evaluation has not been achieved.MethodsWe conducted a qualitative evaluation of the roll-out of the register. This comprised twenty in-depth interviews with a diverse sample of stakeholders at facility, sub-district, and district levels of the health system. Facility-level participants were selected across five sub-districts, including one facility per sub-district. Responses were coded and analysed using a thematic approach. An implementation science framework guided interpretation of the data.ConclusionIn this study we found that relative advantage of an intervention and stakeholder engagement are critical to implementation. We suggest that without these aspects of implementation, formative and summative outcomes of implementation at both the adoption and coalface stages of implementation would be negatively affected.     

Decline of FoxP3+ Regulatory CD4 T Cells in Peripheral Blood of Children Heavily Exposed to Malaria

FoxP3+ regulatory CD4 T cells (T_regs) help to maintain the delicate balance between pathogen-specific immunity and immune-mediated pathology. Prior studies suggest that T_regs are induced by P. falciparum both in vivo and in vitro; however, the factors influencing T_reg homeostasis during acute and chronic infections, and their role in malaria immunopathogenesis, remain unclear. We assessed the frequency and phenotype of T_regs in well-characterized cohorts of children residing in a region of high malaria endemicity in Uganda. We found that both the frequency and absolute numbers of FoxP3+ T_regs in peripheral blood declined markedly with increasing prior malaria incidence. Longitudinal measurements confirmed that this decline occurred only among highly malaria-exposed children. The decline of T_regs from peripheral blood was accompanied by reduced in vitro induction of T_regs by parasite antigen and decreased expression of TNFR2 on T_regs among children who had intense prior exposure to malaria. While T_reg frequencies were not associated with protection from malaria, there was a trend toward reduced risk of symptomatic malaria once infected with P. falciparum among children with lower T_reg frequencies. These data demonstrate that chronic malaria exposure results in altered T_reg homeostasis, which may impact the development of antimalarial immunity in naturally exposed populations.     

Methyl Sulfone Blocked Multiple Hypoxia- and Non-Hypoxia-Induced Metastatic Targets in Breast Cancer Cells and Melanoma Cells

Metastatic cancer causes 90% of cancer deaths. Unlike many primary tumors, metastatic tumors cannot be cured by surgery alone. Metastatic cancer requires chemotherapy. However, metastatic cells are not easily killed by chemotherapy. These problems with chemotherapy are caused in part by the metastatic cell niche: hypoxia. Here we show that the molecule, methyl sulfone, normalized metastatic metabolism of hypoxic breast cancer and melanoma cells by altering several metabolic functions of the cells. Under hypoxia, methyl sulfone decreased expression of the master regulator of hypoxia, HIF-1α, and reduced levels of the glycolytic enzymes, PKM2, LDHA, GLUT1, the pro-angiogenic protein, VEGF, and the iron-sulfur metabolism molecules, miR-210 and transferrin, all of which promote metastasis. Conversely, methyl sulfone increased levels of ISCU1/2 and ferroportin, proteins associated with iron-sulfur cluster biogenesis and iron homeostasis in normal cells. These data identify methyl sulfone as a multi-targeting molecule that blocks the survival/proliferative effect of hypoxia on metastatic cells and brings normality back to cellular metabolism.     

Modeling shifts in the rate and pattern of subthalamopallidal network activity during deep brain stimulation

Deep brain stimulation (DBS) of the subthlamic nucleus (STN) represents an effective treatment for medically refractory Parkinson’s disease; however, understanding of its effects on basal ganglia network activity remains limited. We constructed a computational model of the subthalamopallidal network, trained it to fit in vivo recordings from parkinsonian monkeys, and evaluated its response to STN DBS. The network model was created with synaptically connected single compartment biophysical models of STN and pallidal neurons, and stochastically defined inputs driven by cortical beta rhythms. A least mean square error training algorithm was developed to parameterize network connections and minimize error when compared to experimental spike and burst rates in the parkinsonian condition. The output of the trained network was then compared to experimental data not used in the training process. We found that reducing the influence of the cortical beta input on the model generated activity that agreed well with recordings from normal monkeys. Further, during STN DBS in the parkinsonian condition the simulations reproduced the reduction in GPi bursting found in existing experimental data. The model also provided the opportunity to greatly expand analysis of GPi bursting activity, generating three major predictions. First, its reduction was proportional to the volume of STN activated by DBS. Second, GPi bursting decreased in a stimulation frequency dependent manner, saturating at values consistent with clinically therapeutic DBS. And third, ablating STN neurons, reported to generate similar therapeutic outcomes as STN DBS, also reduced GPi bursting. Our theoretical analysis of stimulation induced network activity suggests that regularization of GPi firing is dependent on the volume of STN tissue activated and a threshold level of burst reduction may be necessary for therapeutic effect.     

Assessment of hydroxypropyl methylcellulose, propylene glycol, polysorbate 80, and hydroxypropyl-β-cyclodextrin for use in developmental and reproductive toxicology studies

BACKGROUND: A series of studies were conducted to assess Polysorbate 80 (PS80), Propylene Glycol (PG), and Hydroxypropyl‐β‐Cyclodextrin (HPβCD), when compared with Hydroxypropyl Methylcellulose (MC) in developmental and reproductive toxicology (DART) studies. METHODS: In the rat fertility study, 20 mg/kg MC, 10 mg/kg PS80, 1,000 mg/kg PG, 500 mg/kg HPβCD or 1,000 mg/kg HPβCD were administered orally before/during mating, and on gestation Day (GD) 0–7, followed by an assessment of embryonic development on GD 14. In the rat and rabbit teratology studies, the doses of MC, PS80, PG, and HPβCD were the same as those in the fertility study. In these teratology studies, pregnant females were dosed during the period of organogenesis, followed by an assessment of fetal external, visceral, and skeletal development. RESULTS: In the rat fertility and rat teratology studies, PS80, PG, and HPβCD did not exhibit toxicity, when compared with MC. Similarly, in the rabbit teratology study, there was no PS80 or PG‐related toxicity, when compared with MC. However, individual rabbits in the 500 and 1,000 mg/kg HPβCD groups exhibited maternal toxicity, which included stool findings, decreased food consumption, and body weight gain. Furthermore, one rabbit each in the 500 and 1,000 mg/kg HPβCD groups exhibited evidence of abortion, which was considered secondary to maternal toxicity. CONCLUSIONS: Although HPβCD was not well tolerated in rabbits at doses of 500 and 1,000 mg/kg, PS80 and PG were comparable to MC and should be considered for use in developmental and reproductive toxicology studies. Birth Defects Res (Part B) 89:504–516,2010. © 2010 Wiley‐Liss, Inc.