Creatine transporter (SLC6A8) knockout mice display an increased capacity for in vitro creatine biosynthesis in skeletal muscle.

The present study aimed to investigate whether skeletal muscle from whole body creatine transporter (CrT; SLC6A8) knockout mice (CrT(-/y)) actually contained creatine (Cr) and if so, whether this Cr could result from an up regulation of muscle Cr biosynthesis. Gastrocnemius muscle from CrT(-/y) and wild type (CrT(+/y)) mice were analyzed for ATP, Cr, Cr phosphate (CrP), and total Cr (TCr) content. Muscle protein and gene expression of the enzymes responsible for Cr biosynthesis L-arginine:glycine amidotransferase (AGAT) and guanidinoacetate methyltransferase (GAMT) were also determined as were the rates of in vitro Cr biosynthesis. CrT(-/y) mice muscle contained measurable (22.3 ± 4.3 mmol.kg(-1) dry mass), but markedly reduced (P < 0.05) TCr levels compared with CrT(+/y) mice (125.0 ± 3.3 mmol.kg(-1) dry mass). AGAT gene and protein expression were higher (~3 fold; P < 0.05) in CrT(-/y) mice muscle, however GAMT gene and protein expression remained unchanged. The in vitro rate of Cr biosynthesis was elevated 1.5 fold (P < 0.05) in CrT(-/y) mice muscle. These data clearly demonstrate that in the absence of CrT protein, skeletal muscle has reduced, but not absent, levels of Cr. This presence of Cr may be at least partly due to an up regulation of muscle Cr biosynthesis as evidenced by an increased AGAT protein expression and in vitro Cr biosynthesis rates in CrT(-/y) mice. Of note, the up regulation of Cr biosynthesis in CrT(-/y) mice muscle was unable to fully restore Cr levels to that found in wild type muscle.

Effects of ATP7A overexpression in mice on copper transport and metabolism in lactation and gestation

Placentae and mammary epithelial cells are unusual in robustly expressing two copper "pumps", ATP7A and B, raising the question of their individual roles in these tissues in pregnancy and lactation. Confocal microscopic evidence locates ATP7A to the fetal side of syncytiotrophoblasts, suggesting a role in pumping Cu towards the fetus; and to the basolateral (blood) side of lactating mammary epithelial cells, suggesting a role in recycling Cu to the blood. We tested these concepts in wild-type C57BL6 mice and their transgenic counterparts that expressed hATP7A at levels 10-20× those of endogenous mAtp7a. In lactation, overexpression of ATP7A reduced the Cu concentrations of the mammary gland and milk ~50%. Rates of transfer of tracer (64)Cu to the suckling pups were similarly reduced over 30-48 h, as was the total Cu in 10-day -old pups. During the early and middle periods of gestation, the transgenic litters had higher Cu concentrations than the wild-type, placental Cu showing the reverse trend; but this difference was lost by the first postnatal day. The transgenic mice expressed ATP7A in some hepatocytes, so we investigated the possibility that metalation of ceruloplasmin (Cp) might be enhanced. Rates of (64)Cu incorporation into Cp, oxidase activity, and ratios of holo to apoceruloplasmin were unchanged. We conclude that in the lactating mammary gland, the role of ATP7A is to return Cu to the blood, while in the placenta it mediates Cu delivery to the fetus and is the rate-limiting step for fetal Cu nutrition during most of gestation in mice.

Impact of introduced house mice (Mus musculus) on burrowing seabirds on Steeple Jason and Grand Jason Islands, Falklands, South Atlantic

Whilst there is good evidence for negative impacts of introduced rat species on island ecosystems, the effects of house mice (Mus musculus) are generally less well documented. In some situations, introduced house mice can exert severe impacts, particularly where this is the only introduced mammal. Here, we examine the distribution, relative abundance and breeding success of small burrowing seabirds on Steeple Jason Island, Falklands, in relation to habitat types and the distribution of house mice which is the sole introduced mammal species, and we make comparisons with seabird distribution and densities on the neighbouring island of Grand Jason where mice are absent. Grey-backed storm-petrel (Garrodia nereis) and Wilson's storm-petrel (Oceanites oceanicus), which due to their extremely small size are likely to be the most vulnerable to mouse predation, were considerably more abundant on mouse-free Grand Jason than on Steeple Jason. Grey-backed storm-petrel, which are typically associated with tussac grass, avoided this habitat on Steeple Jason where it is associated with high levels of house mouse activity (assessed from the proportion of wax baits gnawed overnight), whereas on mouse-free Grand Jason, there was no such avoidance. Wilson's storm-petrel nesting on Steeple Jason suffered high rates of egg and chick loss. Whilst we found evidence for detrimental impacts of house mice on the two small storm-petrel species, there was no relationship between relative mouse activity levels and the distribution or abundance of the larger thin-billed Prion (Pachyptila belcheri). © 2014 Springer-Verlag Berlin Heidelberg.

Foxp3 expression in macrophages associated with RENCA tumors in mice.

The transcription factor Foxp3 represents the most specific functional marker of CD4+ regulatory T cells (TRegs). However, previous reports have described Foxp3 expression in other cell types including some subsets of macrophages, although there are conflicting reports and Foxp3 expression in cells other than Treg is not well characterized. We performed detailed investigations into Foxp3 expression in macrophages in the normal tissue and tumor settings. We detected Foxp3 protein in macrophages infiltrating mouse renal cancer tumors injected subcutaneously or in the kidney. Expression was demonstrated using flow cytometry and Western blot with two individual monoclonal antibodies. Further analyses confirmed Foxp3 expression in macrophages by RT PCR, and studies using ribonucleic acid-sequencing (RNAseq) demonstrated a previously unknown Foxp3 messenger (m)RNA transcript in tumor-associated macrophages. In addition, depletion of Foxp3+ cells using diphtheria toxin in Foxp3DTR mice reduced the frequency of type-2 macrophages (M2) in kidney tumors. Collectively, these results indicate that tumor-associated macrophages could express Foxp3.

Dietary repletion with ω3 fatty acid or with COX inhibition reverses cognitive effects in F3 ω3 fatty-acid-deficient mice

Dietary deficiency of ω3 fatty acid during development leads to impaired cognitive function. However, the effects of multiple generations of ω3 fatty-acid deficiency on cognitive impairment remain unclear. In addition, we sought to test the hypothesis that the cognitive impairments of ω3 fatty-acid-deficient mice are mediated through the arachidonic acid-cyclooxygenase (COX) pathway. To address these issues, C57BL/6J mice were bred for 3 generations and fed diets either deficient (DEF) or sufficient (SUF) in ω3 fatty acids. At postnatal day 21, the F3 offspring remained on the dam's diet or were switched to the opposite diet, creating 4 groups. In addition, 2 groups that remained on the dam's diet were treated with a COX inhibitor. At 19 wk of age, spatial-recognition memory was tested on a Y-maze. Results showed that 16 wk of SUF diet reversed the cognitive impairment of F3 DEF mice. However, 16 wk of ω3 fatty-acid-deficient diet impaired the cognitive performance of the F3 SUF mice, which did not differ from that of the F3 DEF mice. These findings suggest that the cognitive deficits after multigenerational maintenance on ω3 fatty-acid-deficient diet are not any greater than are those after deficiency during a single generation. In addition, treatment with a COX inhibitor prevented spatial-recognition deficits in F3 DEF mice. Therefore, cognitive impairment due to dietary ω3 fatty-acid deficiency appears to be mediated by the arachidonic acid-COX pathway and can be prevented by 16 wk of dietary repletion with ω3 fatty acids or COX inhibition.

Age-related proteostasis and metabolic alterations in Caspase-2-deficient mice.

Ageing is a complex biological process for which underlying biochemical changes are still largely unknown. We performed comparative profiling of the cellular proteome and metabolome to understand the molecular basis of ageing in Caspase-2-deficient (Casp2(-/-)) mice that are a model of premature ageing in the absence of overt disease. Age-related changes were determined in the liver and serum of young (6-9 week) and aged (18-24 month) wild-type and Casp2(-/-) mice. We identified perturbed metabolic pathways, decreased levels of ribosomal and respiratory complex proteins and altered mitochondrial function that contribute to premature ageing in the Casp2(-/-) mice. We show that the metabolic profile changes in the young Casp2(-/-) mice resemble those found in aged wild-type mice. Intriguingly, aged Casp2(-/-) mice were found to have reduced blood glucose and improved glucose tolerance. These results demonstrate an important role for caspase-2 in regulating proteome and metabolome remodelling during ageing.

Sensitivity to MK-801 in phospholipase C-β1 knockout mice reveals a specific NMDA receptor deficit

Phospholipase C-β1 (PLC-β1) is a critical component of multiple signalling pathways downstream of neurotransmitter receptors. Mice lacking this enzyme display a striking behavioural phenotype with relevance to human psychiatric disease. Glutamatergic dysfunction is strongly associated with several abnormal behavioural states and may underlie part of the phenotype of the phospholipase C-β1 knockout (KO) mouse. A heightened response to glutamatergic psychotomimetic drugs is a critical psychosis-related endophenotype, and in this study it was employed as a correlate of glutamatergic dysfunction. Control (n=8) and PLC-β1 KO mice (n=6) were treated with MK-801, a NMDA receptor (NMDAR) antagonist, following either standard housing or environmental enrichment, and the motor function and locomotor activity thus evoked was assessed. In addition, MK-801 binding to the NMDAR was evaluated through radioligand autoradiography in post-mortem tissue (on a drug-naive cohort). We have demonstrated a significantly increased sensitivity to the effects of the NMDA antagonist MK-801 in the PLC-β1 KO mouse. In addition, we found that this mouse line displays reduced hippocampal NMDAR expression, as measured by radioligand binding. We previously documented a reversal of specific phenotypes in this mouse line following housing in an enriched environment. Enrichment did not alter this heightened MK-801 response, nor NMDAR expression, indicating that this therapeutic intervention works on specific pathways only. These findings demonstrate the critical role of the glutamatergic system in the phenotype of the PLC-β1 KO mouse and highlight the role of these interconnected signalling pathways in schizophrenia-like behavioural disruption. These results also shed further light on the capacity of environmental factors to modulate subsets of these phenotypes.

Brain targeted PLGA nanocarriers alleviating amyloid-Β expression and preserving basal survivin in degenerating mice model

The chronic systemic administration of d-Galactose in C57BL/6J mice showed a relatively high oxidative stress, amyloid-β expression and neuronal cell death. Enhanced expression of pyknotic nuclei, caspase-3 and reduced expression of neuronal integrity markers further confirmed the aforesaid insults. However, concomitant treatment with the recombinant protein (SurR9-C84A) and the anti-transferrin receptor antibody conjugated SurR9-C84A (SurR9+TFN) nanocarriers showed a significant improvement in the disease status and neuronal health. The beauty of this study is that the biodegradable Food and Drug Administration (FDA) approved poly(lactic-co-glycolic acid) (PLGA) nanocarriers enhanced the biological half-life and the efficacy of the treatments. The nanocarriers were effective in lowering the amyloid-β expression, enhancing the neuronal integrity markers and maintaining the basal levels of endogenous survivin that is essential for evading the caspase activation and apoptosis. The current study herein reports for the first time that the brain targeted SurR9-C84A nanocarriers alleviated the d-Galactose induced neuronal insults and has potential for future brain targeted nanomedicine application.

Antimanic-like activity of candesartan in mice: possible involvement of antioxidant, anti-inflammatory and neurotrophic mechanisms

Activation of the brain angiotensin II type 1 receptor (AT1R) triggers pro-oxidant and pro-inflammatory mechanisms which are involved in the neurobiology of bipolar disorder (BD). Candesartan (CDS) is an AT1 receptor antagonist with potential neuroprotective properties. Herein we investigated CDS effects against oxidative, neurotrophic inflammatory and cognitive effects of amphetamine (AMPH)-induced mania. In the reversal protocol adult mice were given AMPH 2mg/kg i.p. or saline and between days 8 and 14 received CDS 0.1, 0.3 or 1mg/kg orally, lithium (Li) 47.5mg/kg i.p., or saline. In the prevention treatment, mice were pretreated with CDS, Li or saline prior to AMPH. Locomotor activity and working memory performance were assessed. Glutathione (GSH), thiobarbituric acid-reactive substance (TBARS) and TNF-α levels were evaluated in the hippocampus (HC) and cerebellar vermis (CV). Brain-derived neurotrophic factor (BDNF) and glycogen synthase kinase 3-beta (GSK-3beta) levels were measured in the HC. CDS and Li prevented and reversed the AMPH-induced increases in locomotor activity. Only CDS prevented and reversed AMPH-induced working memory deficits. CDS prevented AMPH-induced alterations in GSH (HC and CV), TBARS (HC and CV), TNF-α (HC and CV) and BDNF (HC) levels. Li prevented alterations in BDNF and phospho-Ser9-GSK3beta. CDS reversed AMPH-induced alterations in GSH (HC and CV), TBARS (HC), TNF-α (CV) and BDNF levels. Li reversed AMPH-induced alterations in TNF-α (HC and CV) and BDNF (HC) levels. CDS is effective in reversing and preventing AMPH-induced behavioral and biochemical alterations, providing a rationale for the design of clinical trials investigating CDS׳s possible therapeutic effects.

The PTEX component EXP2 is critical for establishing a patent malaria infection in mice

Export of most malaria proteins into the erythrocyte cytosol requires the Plasmodium Translocon of Exported proteins (PTEX) and a cleavable Plasmodium Export Element (PEXEL). In contrast, the contribution of PTEX in the liver stages and export of liver stage proteins is unknown. Here, using the FLP/FRT conditional mutatagenesis system, we generate transgenic P. berghei parasites deficient in EXP2, the putative pore-forming component of PTEX. Our data reveal that EXP2 is important for parasite growth in the liver and critical for parasite transition to the blood, with parasites impaired in their ability to generate a patent blood-stage infection. Surprisingly, whilst parasites expressing a functional PTEX machinery can efficiently export a PEXEL-bearing GFP reporter into the erythrocyte cytosol during a blood stage infection, this same reporter aggregates in large accumulations within the confines of the parasitophorous vacuole membrane during hepatocyte growth. Notably HSP101, the putative molecular motor of PTEX, could not be detected during the early liver stages of infection, which may explain why direct protein translocation of this soluble PEXEL-bearing reporter or indeed native PEXEL proteins into the hepatocyte cytosol has not been observed. This suggests that PTEX function may not be conserved between the blood and liver stages of malaria infection. This article is protected by copyright. All rights reserved.