Decrease In Basal Levels Research Articles

ABCG2 and SLC1A5 functionally interact to rewire metabolism and confer a survival advantage to cancer cells under oxidative stress

ABCG2, a member of the ABC transporter superfamily, is overexpressed in many human tumors and has long been studied for its ability to export a variety of chemotherapeutic agents, thereby conferring a multidrug resistance (MDR) phenotype. However, several studies have shown that ABCG2 can also confer an MDR-independent survival advantage to tumor cells exposed to stress. While investigating the mechanism by which ABCG2 enhances survival in stressful milieus, we have identified a physical and functional interaction between ABCG2 and SLC1A5, a member of the solute transporter superfamily and the primary transporter of glutamine in cancer cells. This interaction was accompanied by increased glutamine uptake, increased glutaminolysis and rewired cellular metabolism, as evidenced by an increase in key metabolic enzymes and alteration of glutamine-dependent metabolic pathways. Specifically, we observed an increase in glutamine metabolites shuttled to the TCA cycle, an increase in the synthesis of glutathione, accompanied by a decrease in basal levels of reactive oxygen species and a marked increase in cell survival in the face of oxidative stress. Notably, knockdown of SLC1A5 or depletion of exogenous glutamine diminished ABCG2-enhanced autophagy flux, further implicating this solute transporter in ABCG2-mediated cell survival. This is, to our knowledge, the first report of a functionally significant physical interaction between members of the two major transporter superfamilies. Moreover, these observations may underlie the protective role of ABCG2 in cancer cells under duress and suggest a novel role for ABCG2 in the regulation of metabolism in normal and diseased states.

Absence of Bcl11b in murine heart leads to mitochondrial dysfunction and cardiomyocyte death

B cell lymphoma 11b (Bcl11b/Ctip2) is a zinc finger transcriptional factor involved in a number of physiological processes, including cardiac hypertrophy. In humans, genome-wide association studies show that a common genetic variation in the 3’ region of Bcl11b gene is associated with higher risk of aortic stiffness and increased rate of cardiovascular disease. In mice, Bcl11b invalidation specifically in cardiomyocytes leads to cardiac malfunction, fibrosis augmentation and cell death. Our specific aims are: To investigate the signalling pathways related to cardiac cell death and to assess the sarcomere structure of cardiomyocytes in the absence of Bcl11b. The experiments were conducted using cardiac specific Bcl11b knock-out mice induced by tamoxifen administration 3 and 7 days after the invalidation, cardiac tissues were collected to perform RNA sequencing and subsequently to analyse related molecular pathways using bioinformatic tools. In parallel, cardiomyocytes were purified and isolated by conducting Langendorff perfusion technique to have a better insight into the impact of Bcl11b. A simplified mathematical model, Fourier transform analysis was used to assess sarcomeric length following Bcl11b invalidation in cardiomyocytes. Based on the RNA sequencing results, Bcl11b invalidation has an impact on the regulation of over 2000 genes. Among them, genes related to cell death and mitochondrial functions are the most affected regarding the enrichment scores. Interestingly, a considerable number of genes fall into the “Cuproptosis” pathway, a newly identified, non-apoptotic cell death mechanism. The oxygen consumption of mitochondria was also measured using the Seahorse assay. Bcl11b deletion leads to the decrease in basal level of mitochondrial respiration with no effect on the maximum respiratory capacity. Finally, adult cardiomyocytes in the mutant groups showed a moderate hypertrophy (15% bigger than the control group). However, Fourier transform analysis exhibited stability in the average sarcomere length of these cells. The obtained data suggest that cardiac specific invalidation of Bcl11b induces mitochondrial dysfunction and possibly cell death through the “Cuproptosis” pathway.

Hyperbaric oxygenation applied before or after mild or hard stress: effects on the redox state in the muscle tissue.

The mechanism is unclear for the reported protective effect of hyperbaric oxygen preconditioning against oxidative stress in tissues, and the distinct effects of hyperbaric oxygen applied after stress. The trained mice were divided into three groups: the control, hyperbaric oxygenation preconditioning, and hyperbaric oxygenation applied after mild (fasting) or hard (prolonged exercise) stress. After preconditioning, we observed a decrease in basal levels of nitric oxide, tetrahydrobiopterin, and catalase despite the drastic increase in inducible and endothelial nitric oxide synthases. Moreover, the basal levels of glutathione, related enzymes, and nitrosative stress only increased in the preconditioning group. The control and preconditioning groups showed a similar mild stress response of the endothelial and neuronal nitric oxide synthases. At the same time, the activity of all nitric oxide synthase, glutathione (GSH) in muscle, declined in the experimental groups but increased in control during hard stress. The results suggested that hyperbaric oxygen preconditioning provoked uncoupling of nitric oxide synthases and the elevated levels of GSH in muscle during this study, while hyperbaric oxygen applied after stress showed a lower level of GSH but higher recovery post-exercise levels in the majority of antioxidant enzymes. We discuss the possible mechanisms of the redox response and the role of the nitric oxide in this process.

Expression Profile of Genes Related to the Th17 Pathway in Macrophages Infected by Leishmania major and Leishmania amazonensis: The Use of Gene Regulatory Networks in Modeling This Pathway.

Leishmania amazonensis and Leishmania major are the causative agents of cutaneous and mucocutaneous diseases. The infections‘ outcome depends on host–parasite interactions and Th1/Th2 response, and in cutaneous form, regulation of Th17 cytokines has been reported to maintain inflammation in lesions. Despite that, the Th17 regulatory scenario remains unclear. With the aim to gain a better understanding of the transcription factors (TFs) and genes involved in Th17 induction, in this study, the role of inducing factors of the Th17 pathway in Leishmania–macrophage infection was addressed through computational modeling of gene regulatory networks (GRNs). The Th17 GRN modeling integrated experimentally validated data available in the literature and gene expression data from a time-series RNA-seq experiment (4, 24, 48, and 72 h post-infection). The generated model comprises a total of 10 TFs, 22 coding genes, and 16 cytokines related to the Th17 immune modulation. Addressing the Th17 induction in infected and uninfected macrophages, an increase of 2- to 3-fold in 4–24 h was observed in the former. However, there was a decrease in basal levels at 48–72 h for both groups. In order to evaluate the possible outcomes triggered by GRN component modulation in the Th17 pathway. The generated GRN models promoted an integrative and dynamic view of Leishmania–macrophage interaction over time that extends beyond the analysis of single-gene expression.

Нарушения репродуктивной функции и гормонального профиля у пациенток с АКТГ-секретирующими аденомами гипофиза

Актуальность. Синдром Кушинга (СК) — тяжелая нейроэндокринная патология, которая уже многие годы находится под пристальным вниманием различных специалистов вследствие разноплановости ее осложнений. Гиперандрогения и нарушения менструального цикла, такие как аменорея и олигоменорея, дополняют клиническую картину СК. Цель исследования: оценить состояние фертильной функции и гормонального профиля у пациенток с синдромом Кушинга. Материалы и методы. Под наблюдением по поводу СК находились в период с 2000 по 2019 г. 163 пациентки фертильного возраста. Средний возраст обследуемых составил 27,58 ± 3,40 года (от 17 до 49 лет), длительность заболевания — 4,2 ± 0,2 года. 20 здоровых женщин соответствующего возраста составили группу контроля. Результаты. Пациентки с СК были распределены следующим образом: первая группа — с АКТГ-зависимым СК — 130 (79,7 %), вторая группа — с АКТГ-независимым СК — 30 (18,4 %) и третья группа — с АКТГ-эктопированным СК — 3 (1,8 %). Среди различных репродуктивных нарушений у пациенток с СК преобладали нарушения менструального цикла и вторичная аменорея. Для пациенток первой группы на 14-й день менструального цикла характерным было достоверное снижение базальных уровней лютеинизирующего и фолликулостимулирующего гормонов, снижение уровня овариальных гормонов (эстрадиол, прогестерон) на фоне гиперандрогенемии и достоверное повышение уровня кортизола крови при увеличении показателя АКТГ. Гиперплазия обоих надпочечников диагностирована у 110 (84,6 %) больных с АКТГ-зависимым СК, узелковая гиперплазия обоих надпочечников — у 13 (10 %), инциденталома — у 3 (2,3 %), узелковая гиперплазия одного надпочечника — у 2 (1,5 %). Выводы. У пациенток с синдромом Кушинга наблюдались репродуктивные расстройства: нарушение менструального цикла — у 32,9 %, вторичная аменорея — у 20,4 %. Большая часть нарушений менструального цикла установлена в группе больных с АКТГ-зависимым синдромом Кушинга. Нарушения менструального цикла проявлялись в виде олигоменореи (30 % случаев), аменореи (35 %), полименореи (7,5 %) и вариабельности цикла (7,5 %).

Study of the vasodilatory effect of extract from tissue culture biomass of Rauwolfia serpentina Benth.

Design of novel herbal drugs with a wide range of activity is an important object of pharmacology. Herbal remedies having antiarrhythmic, cardiotonic and vasodilatory effects are useful for treatment of heart arrhythmias. Vasodilating drugs are prescribed for medication of hypertension and migraine. Also they are a part of comprehensive treatment of various diseases such as disturbed peripheral blood circulation and atherosclerosis of arteries of extremities as well as problems with urination and potency. Search for novel herbal preparations with vasorelaxant activity in our view is a promising matter of current interest.
 This work is aiming in study of vasodilatory activity of methanol extract of strain K-27 tissue culture of Rauwolfia serpentina.
 Evaluation of vasodilatory activity extract of high-productive strain K-27 tissue culture of Rauwolfia serpentina has been carried out by the method of auxotonic mechanography of vascular smooth muscles. Strength and frequency of spontaneous contractions of portal vein as well as relaxation of aortic smooth muscles activated with phenylephrine were evaluation indicators of extract activity.
 In order to estimate working concentration of extract of strain K-27 tissue culture of Rauwolfia serpentina we have studied vascular activity at sequentially increasing concentrations of extract solution on the portal vein. Overlay of sequence of growing concentrations exhibited decrease in basal level of venous tone and dose-dependent suppression of phasic contractions with complete inhibition of spontaneous activity of portal vein at 1.44 mg/ml concentration and uprise of toxicity at higher concentrations. It has been shown dose-dependent relaxation of vascular preparations followed by development of α-adrenoceptor blocking effect and loss of sensibility to phenylephrine at the end of experiment. After discontinuing exposure with a substance the activating effect of phenylephrine remained not renewed during 30-50 minutes, at the same time contractile activity of aorta on the response to the other type activation (60 mM K+) took place.
 It was found that at the studied range of concentrations: 0.0288 μg/ml–28.8 μg/ml the extract of strain K-27 tissue culture of Rauwolfia serpentina has distinct vasodilatory effect. Also it was discovered α-adrenoceptor blocking effect of that extract, so it can find wide application in therapy as vasorelaxant and α-blocker, for instance in treatment of prostate diseases.

Abrogation of LRRK2 dependent Rab10 phosphorylation with TLR4 activation and alterations in evoked cytokine release in immune cells

LRRK2 protein is expressed prominently in immune cells, cell types whose contribution to LRRK2-associated genetic Parkinson's disease (PD) is increasingly being recognised. We investigated the effect of inflammatory stimuli using RAW264.7 murine macrophage cells as model systems. A detailed time course of TLR2 and TLR4 stimulation was investigated through measuring LRRK2 phosphorylation at its specific phospho-sites, and Rab8 and Rab10 phosphorylation together with cytokine release following treatment with LPS and zymosan. LRRK2 phosphorylation at Ser935, Ser955 and Ser973 was increased significantly over untreated conditions at 4–24h in both WT-LRRK2 and T1348N-LRRK2 cell lines to similar extents although levels of Ser910 phosphorylation were maintained at higher levels throughout. Importantly we demonstrate that LPS stimulation significantly decreased phospho-Rab10 but not phospho-Rab8 levels over 4–24h in both WT-LRRK2 and T1348N-LRRK2 cell lines. The dephosphorylation of Rab10 was not attributed to its specific phosphatase, PPM1H as the levels remained unaltered with LPS treatment. MAPK phosphorylation occurred prior to LRRK2 phosphorylation which was validated by blocking TLR4 and TLR2 receptors with TAK242 or Sparstolonin B respectively. A significant decrease in basal level of TNFα release was noted in both T1348N-LRRK2 and KO-LRRK2 cell lines at 48h compared to WT-LRRK2 cell line, however LPS and zymosan treatment did not cause any significant alteration in the TNFα and IL-6 release between the three cell lines. In contrast, LPS and zymosan caused significantly lower IL-10 release in T1348N-LRRK2 and KO-LRRK2 cell lines. A significant decrease in phospho-Rab10 levels was also confirmed in human IPS-derived macrophages with TLR4 activation. Our data demonstrates for the first time that LRRK2-dependent Rab10 phosphorylation is modulated by LPS stimulation, and that cytokine release may be influenced by the status of LRRK2. These data provide further insights into the function of LRRK2 in immune response, and has relevance for understanding cellular dysfunctions when developing LRRK2-based inhibitors for clinical treatment.

Клініко-діагностичні аспекти дефіциту тестостерону в жінок фертильного віку

Актуальність. До сьогодні роль андрогенів в організмі жінки не отримала належної оцінки. Поки не існує чіткого і загальноприйнятого визначення поняття «андрогенний дефіцит у жінок» за аналогією з таким у чоловіків. Мета дослідження: вивчити клініко-діагностичні аспекти дефіциту тестостерону в жінок репродуктивного віку. Матеріали та методи. Під наглядом перебувало 60 жінок із дефіцитом тестостерону без супутньої патології. Середній вік пацієнток становив 28,3 року. Контрольну групу становили 20 здорових жінок. Пацієнтки були розподілені за показником індексу маси тіла (ІМТ) на три групи. Першу групу становили 20 жінок із надмірною масою тіла (ІМТ 25–29,9 кг/м2), другу групу— 20 жінок з ожирінням I ступеня і третю групу— 20 жінок з ожирінням II ступеня. Результати. Для пацієнток із групи з ожирінням I ступеня була характерна на 14-й день оваріально-менструального циклу тенденція до зниження базальних рівнів лютеїнізуючого гормона (ЛГ), фолікулостимулюючого гормона (ФСГ), естрадіолу і прогестерону на тлі вірогідної гіпоандрогенемії. У пацієнток із групи з ожирінням II ступеня відзначалося вірогідне зниження базальних рівнів ЛГ, ФСГ, естрадіолу, прогестерону на тлі вірогідної гіпоандрогенемії. При проведенні кореляційного аналізу виявлений прямий зв’язок дефіциту тестостерону зі ступенем ожиріння. Так, найнижчі значення тестостерону виявлені в пацієнток третьої групи (<0,05нмоль/л). Водночас у пацієнток першої і другої груп рівень тестостерону також був значно знижений— від 0,02 до 0,05 нмоль/л відповідно. Вірогідно зниженими виявилися рівні дегідроепіандростерону и 17-оксипрогестерону, особливо в третій групі— 0,3 мкг/мл і 0,1 нмоль/л відповідно. Висновки. У більшості жінок репродуктивного віку з дефіцитом тестостерону спостерігається гіпоменорея з низьким вмістом вільного тестостерону. Встановлено пряму кореляційну залежність дефіциту тестостерону від ступеня ожиріння в жінок з андрогенною недостатністю.

542 Xeroderma Pigmentosum C mutations increase oxidative DNA damages by inhibiting Base Excision Repair System’s expression and activity

Xeroderma Pigmentosum C (XPC) is a rare genodermatosis which manifests clinically as pronounced photosensitivity, abnormal pigmentation and numerous skin cancers. It is caused by mutations in XPC gene that initiates global-genome nucleotide excision repair (GG-NER), a repair system responsible for bulky DNA adducts removal. Interestingly, development of internal and skin cancers in non-photo-exposed areas in parallel to an increase in oxidative DNA damages are observed upon these mutations. Therefore, we propose in our work to study XPC role in the modulation of BER, a major route for repairing oxidative DNA damage, at transcriptomic and proteomic levels along with repair activity using normal and XPC-mutated primary fibroblasts. To do so we used real time q-PCR and western blot to check effect of different XPC mutations on BER enzymes (OGG1, MYH in particular) at basal level and upon ultraviolet radiation type B (UVB) stress. Meanwhile, for studying the effect on BER’s activity, we measured the kinetic of repair of 7, 8-dihydro-8-oxoguanine (8-oxoguanine) using comet assay associated with formamidopyrimidine glycosylase (Fpg). A decrease in basal level and stress-induced level of BER was observed at gene and protein expression levels. Interestingly OGG1 and MYH, both of which are involved in 8-oxoguanine repair, were significantly downregulated. Finally, upon comet-fpg, a higher prolonged 8-oxoguanine oxidative lesions, persisting for more than 24 hours, were seen in XPC-mutated fibroblasts compared to the control especially after UVB-stress. Upon this, XPC deficient cell lines exhibit a reduction in 8-oxoguanine excision capacity, in agreement with the reduced genomic and proteomic levels BER enzymes. This may explain the higher incidence of skin cancer, especially in non-photo-exposed areas where impaired BER leads to an accumulation of lesions in the genome upon an increased induction and poorer repair of oxidative lesions.

Hypoxia alters the expression of hif-1a mRNA and downstream HIF-1 response genes in embryonic and larval lake whitefish (Coregonus clupeaformis)

Lake whitefish (Coregonus clupeaformis) embryos and larvae were exposed to hypoxia at different developmental ages to determine when the cellular response to hypoxia could be initiated. mRNA levels of hypoxia-inducible factor 1α (hif-1α), hsp70, and several HIF-1 target genes were quantified in embryos at 21, 38, 63, 83- and 103-days post fertilisation (dpf) and in larvae at 1, 2, 3- and 4-weeks post hatch (wph) following a 6-hour hypoxia exposure. hsp70 mRNA levels were increased in response to hypoxia at all embryonic ages. By comparison, the first observed change in hif-1α mRNA in response to hypoxia was at 38 dpf, where it was down-regulated from high basal levels, with this response persisting through to 83 dpf. Interestingly, this decrease in hif-1α mRNA coincided with increases in the mRNA levels of the HIF-1 target genes: vegfa (vascular endothelial growth factor A), igfbp1 (insulin-like growth factor binding protein 1), ldha (lactate dehydrogenase a), gapdh (glyceraldehyde-3-phosphate dehydrogenase) and epo (erythropoietin) at select ages. Collectively, this suggests a possible HIF-1-mediated response to hypoxia despite a decrease in hif-1α mRNA. Coinciding with a decrease in basal levels, increases in hif-1α were measured in response to hypoxia at 103 dpf and in larval fish at 1, 2 and 3 wph but there were no consistent increases in HIF-1 target genes at these ages. Overall, our findings indicate that lake whitefish can mount a response to hypoxia early in embryogenesis which may mitigate some of the damaging effects of exposure to low oxygen levels at these critical life history stages.

Direct anti-inflammatory effects of angiotensin-(1-7) on microglia.

Much evidence indicates that pro-inflammatory effects of the renin-angiotensin system within the hypothalamus, including microglial activation and production of pro-inflammatory cytokines, play a role in chronic neurogenic hypertension. Our objective here was to examine whether angiotensin-(1-7) [Ang-(1-7)], a protective component of the renin-angiotensin system, exerts direct actions at microglia to counteract these pro-inflammatory effects. Mas, the Ang-(1-7) receptor, was shown to be present on cultured hypothalamic microglia. Treatment of these cells with Ang-(1-7) (100-1000 nM, 3-12 h) elicited significant decreases in basal levels of mRNAs for the pro-inflammatory cytokines interleukin-1β (IL-1β) and tumor-necrosis factor α (TNFα) and of the microglia-macrophage marker CD11b, and increases in basal levels of the anti-inflammatory cytokine interleukin-10. Incubation of microglial cultures with (pro)renin (PRO) (10-50 nM; 6 h) elicited significant increases in mRNAs for IL-1β, TNFα and CD11b. The effects of PRO (10 nM) on IL-1β and TNFα mRNAs, and TNFα protein, were significantly attenuated by co-treatment with Ang-(1-7) (100 nM). Lastly, these actions of Ang-(1-7) were abolished by the Mas antagonist A-779, and were associated with reductions in NF-κB subunit expression. Collectively, these data provide the first evidence that Ang-(1-7) can exert direct effects at microglia to lower baseline and counteract PRO-induced increases in pro-inflammatory cytokines. Renin-Angiotensin system mediated microglial activation and pro-inflammatory cytokine production within the hypothalamus are components of the chronic neuroinflammation associated with 'neurogenic' hypertension. We demonstrated that angiotension-(1-7) acting via its receptor Mas on hypothalamic microglia lessens baseline and (pro)renin-induced increases in pro-inflammatory cytokine production by these cells. This is the first evidence that angiotensin-(1-7) has direct anti-inflammatory effects via microglia.

Characterization of Novel Src Family Kinase Inhibitors to Attenuate Microgliosis

Microgliosis is a major hallmark of Alzheimer’s disease (AD) brain pathology. Aβ peptide is hypothesized to act as a stimulus for microglia leading to activation of non-receptor tyrosine kinases and subsequent secretion of pro-inflammatory cytokines. Therefore, the signaling pathways mediating microglial activation may be important therapeutic targets of anti-inflammatory therapy for AD. Four novel compounds were chosen after high throughput screening kinase activity assays determined them as potential Lyn kinase inhibitors. Their kinase inhibitory and anti-inflammatory effect on Aβ-stimulated activation was assessed using the murine microglial cell line, BV2. Cells were treated with the compounds to determine effects on active, phosphorylated levels of Src family kinases, Src and Lyn, as well as MAP kinases ERK, JNK and p38. Only one compound, LDDN-0003499, produced a dose dependent decrease in basal levels of active, phosphorylated Src and Lyn in the BV2 cells. LDDN-0003499 treatment also attenuated the Aβ-stimulated increase in active, phosphorylated levels of Lyn/Src and TNFα and IL-6 secretion. This study identifies a novel small molecule Src family tyrosine kinase inhibitor with anti-inflammatory effects in response to Aβ stimulation of microglia. Further in vitro/in vivo characterization of LDDN-0003499 as well as structural modification may provide a new tool for attenuating microglial-mediated brain inflammatory conditions such as that occurring in AD.

Alzheimer’s Disease-Associated Neurotoxic Peptide Amyloid-Β Impairs Base Excision Repair in Human Neuroblastoma Cells

Alzheimer’s disease (AD) is the leading cause of dementia in developed countries. It is characterized by two major pathological hallmarks, one of which is the extracellular aggregation of the neurotoxic peptide amyloid-β (Aβ), which is known to generate oxidative stress. In this study, we showed that the presence of Aβ in a neuroblastoma cell line led to an increase in both nuclear and mitochondrial DNA damage. Unexpectedly, a concomitant decrease in basal level of base excision repair, a major route for repairing oxidative DNA damage, was observed at the levels of both gene expression and protein activity. Moreover, the addition of copper sulfate or hydrogen peroxide, used to mimic the oxidative stress observed in AD-affected brains, potentiates Aβ-mediated perturbation of DNA damage/repair systems in the “Aβ cell line”. Taken together, these findings indicate that Aβ could act as double-edged sword by both increasing oxidative nuclear/mitochondrial damage and preventing its repair. The synergistic effects of increased ROS production, accumulated DNA damage and impaired DNA repair could participate in, and partly explain, the massive loss of neurons observed in Alzheimer’s disease since both oxidative stress and DNA damage can trigger apoptosis.

Characterising an aromatase inhibitor resistant breast cancer cell line

Results Let-R cells have higher levels of ERa compared to Aros. CyclinD1 basal levels are elevated in Aro cells compared to Let-Rs. Levels of JNK and c-jun are higher in Let-R cells compared to Aros. A highly effective ERa knockdown in Let-R cells was optimized. Conclusions The significant ERa knockdown achieved in the Let-R cells will enable further examination of knockdown effects on protein levels and mRNA expression. Elevated expression of ERa in the Let-R cells observed is consistent with the association between AI resistance and ER hypersensitivity. In resistant cells, the decrease in basal levels of CyclinD1 may be partly responsible for the lack of regulation of classical genes like pS2, previously detected by the lab. The elevated levels of JNK and c-jun observed in Let-R cells supports the theory of crosstalk between the ER and growth factor pathways in resistance. Furthermore, it would be of interest to accompany AI treatments with growth factor inhibitors to further our understanding of endocrine resistance.

Is the Selective Serotonin Reuptake Inhibitor Fluoxetine a Fountain of Youth?

Evaluation of: Maya Vetencourt JF, Sale A, Viegi A et al.: The antidepressant fluoxetine restores plasticity in the adult visual cortex. Science 320, 385–288 (2008). The potential for synaptic strength to be regulated by experience is high early in postnatal life, and declines thereafter. The constraint of synaptic plasticity in the adult visual system is thought to underlie the resistance to therapy for the treatment of amblyopia. However, a recent report by Maya Vetencourt et al. demonstrates that systemic administration of the popular antidepressant fluoxetine reactivates synaptic plasticity in the visual system of adult rats, and enables the recovery of function in an eye chronically deprived of vision. Fluoxetine administration stimulated a decrease in basal levels of extracellular GABA, an increase in the level of BDNF and the return of long-term potentiation in slices of visual cortex. The ability to reactivate robust synaptic plasticity in the adult mammalian visual system has clear therapeutic potential for the treatment of amblyopia in adults.

Muscle-Specific Deletion of Rictor Impairs Insulin-Stimulated Glucose Transport and Enhances Basal Glycogen Synthase Activity

Rictor is an essential component of mTOR (mammalian target of rapamycin) complex 2 (mTORC2), a kinase complex that phosphorylates Akt at Ser473 upon activation of phosphatidylinositol 3-kinase (PI-3 kinase). Since little is known about the role of either rictor or mTORC2 in PI-3 kinase-mediated physiological processes in adult animals, we generated muscle-specific rictor knockout mice. Muscle from male rictor knockout mice exhibited decreased insulin-stimulated glucose uptake, and the mice showed glucose intolerance. In muscle lacking rictor, the phosphorylation of Akt at Ser473 was reduced dramatically in response to insulin. Furthermore, insulin-stimulated phosphorylation of the Akt substrate AS160 at Thr642 was reduced in rictor knockout muscle, indicating a defect in insulin signaling to stimulate glucose transport. However, the phosphorylation of Akt at Thr308 was normal and sufficient to mediate the phosphorylation of glycogen synthase kinase 3 (GSK-3). Basal glycogen synthase activity in muscle lacking rictor was increased to that of insulin-stimulated controls. Consistent with this, we observed a decrease in basal levels of phosphorylated glycogen synthase at a GSK-3/protein phosphatase 1 (PP1)-regulated site in rictor knockout muscle. This change in glycogen synthase phosphorylation was associated with an increase in the catalytic activity of glycogen-associated PP1 but not increased GSK-3 inactivation. Thus, rictor in muscle tissue contributes to glucose homeostasis by positively regulating insulin-stimulated glucose uptake and negatively regulating basal glycogen synthase activity.

Aging-Dependent Changes in the Radiation Response of the Adult Rat Brain

To assess the impact of aging on the radiation response in the adult rat brain. Male rats 8, 18, or 28 months of age received a single 10-Gy dose of whole-brain irradiation (WBI). The hippocampal dentate gyrus was analyzed 1 and 10 weeks later for sensitive neurobiologic markers associated with radiation-induced damage: changes in density of proliferating cells, immature neurons, total microglia, and activated microglia. A significant decrease in basal levels of proliferating cells and immature neurons and increased microglial activation occurred with normal aging. The WBI induced a transient increase in proliferation that was greater in older animals. This proliferation response did not increase the number of immature neurons, which decreased after WBI in young rats, but not in old rats. Total microglial numbers decreased after WBI at all ages, but microglial activation increased markedly, particularly in older animals. Age is an important factor to consider when investigating the radiation response of the brain. In contrast to young adults, older rats show no sustained decrease in number of immature neurons after WBI, but have a greater inflammatory response. The latter may have an enhanced role in the development of radiation-induced cognitive dysfunction in older individuals.

M3 Muscarinic Receptor Antagonists Inhibit Small Cell Lung Carcinoma Growth and Mitogen-Activated Protein Kinase Phosphorylation Induced by Acetylcholine Secretion

The importance of acetylcholine as a neurotransmitter in the nervous system is well established, but little is yet known about its recently described role as an autocrine and paracrine hormone in a wide variety of nonneuronal cells. Consistent with the expression of acetylcholine in normal lung, small cell lung carcinoma (SCLC) synthesize and secrete acetylcholine, which acts as an autocrine growth factor through both nicotinic and muscarinic cholinergic mechanisms. The purpose of this study was to determine if interruption of autocrine muscarinic cholinergic signaling has potential to inhibit SCLC growth. Muscarinic receptor (mAChR) agonists caused concentration-dependent increases in intracellular calcium and mitogen-activated protein kinase (MAPK) and Akt phosphorylation in SCLC cell lines. The inhibitory potency of mAChR subtype-selective antagonists and small interfering RNAs (siRNAs) on acetylcholine-increased intracellular calcium and MAPK and Akt phosphorylation was consistent with mediation by M3 mAChR (M3R). Consistent with autocrine acetylcholine secretion stimulating MAPK and Akt phosphorylation, M3R antagonists and M3R siRNAs alone also caused a decrease in basal levels of MAPK and Akt phosphorylation in SCLC cell lines. Treatment of SCLC cells with M3R antagonists inhibited cell growth both in vitro and in vivo and also decreased MAPK phosphorylation in tumors in nude mice in vivo. Immunohistochemical staining of SCLC and additional cancer types showed frequent coexpression of acetylcholine and M3R. These findings suggest that M3R antagonists may be useful adjuvants for treatment of SCLC and, potentially, other cancers.

Age-Related Changes in Adrenomedullin Expression and Hypoxia-Inducible Factor-1 Activity in the Rat Lung and Their Responses to Hypoxia

Male rats aged 3 months, 12 months and 20 months were subjected to breathing 8% oxygen for 6 hours. Lung preproadrenomedullin (AM) messenger RNA (mRNA) levels were measured by solution hybridization-RNase protection assay while AM was measured by radioimmunoassay. The binding of hypoxia-inducible factor-1alpha (HIF-1alpha) to DNA was determined by electrophoretic mobility shift. There was an age-related increase in basal levels of preproAM mRNA and AM and of the binding of hypoxia-inducible factor (HIF) to DNA. Upon hypoxic stimulation, HIF binding to DNA increased in the young and middle-aged rats, but not in the old rats. AM gene expression increased in response to hypoxia in rats of all ages, but the increase was much less in the old rats. AM peptide levels in the lung decreased with age in hypoxia. In a separate experiment, male rats aged 3 months and 20 months were subjected to hypoxia as described above. PreproAM, calcitonin receptor-like receptor (CRLR), receptor activity modifying protein (RAMP) mRNA, HIF-1 and peptidyl-glycine-amidating monooxygenase (PAM) mRNA levels were measured by reverse transcription-polymerase chain reaction. All except PAM showed a decrease in basal levels and a diminished response to hypoxia in the old rats. Polysome profiling demonstrated decreases in the percentages of translatable preproAM mRNA in response to hypoxia, with a greater decrease in the old than the young rats. It is concluded that an age-dependent decrease in the hypoxic response of the AM system in the lung was associated with high basal levels of HIF activity and AM expression in the old rats, and a lower proportion of translatable preproAM mRNA in the old rats in response to hypoxia. Thus, the HIF-AM pathway may be impaired in the aged lung, and other mechanisms may be present to maintain an AM response to hypoxia.

Requirement for Akt-mediated survival in cell transformation by the dbl oncogene

The dbl oncogene product is the founding member of a large family of oncogenic proteins that function by activating the small GTP-binding proteins Cdc42, Rac and Rho. Through its substrate GTPases, Dbl transduces proliferative signals from cell-surface receptors to diverse cellular effectors and signaling pathways. The mechanisms by which these multiple signals are integrated, as well as their relative contribution to Dbl-induced cell transformation, are presently poorly understood. We investigated the role of the survival regulators PI3-kinase and Akt in Dbl-induced cell transformation. We found that Dbl induced the phosphorylation of Akt on threonine 308, through the GTPases Rac and Cdc42 and in a PI3-kinase dependent manner. Pharmacological or biochemical interference with this pathway lead to a marked, dose-dependent inhibition of the focus formation activity exhibited by Dbl-expressing cells. Dbl expression stimulated the phosphorylation of the anti-apoptotic Akt substrate Bad, and caused a marked decrease in basal levels of apoptosis. Finally, we found that activated Cdc42 existed in cells in complex with phosphoionositide-dependent kinase-1 (PDK1), the downstream mediator of PI3-kinase action. The data indicate that Dbl signaling stimulate the formation of a novel survival complex, through which anti-apoptotic signals are generated and propagated.