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Anti-N-methyl-d-aspartate receptor (NMDAR) encephalitis is an immune-mediated disease characterized by a complex neuropsychiatric syndrome in association with an antibody-mediated decrease of NMDAR. About 85% of patients respond to immunotherapy (and removal of an associated tumour if it applies), but it often takes several months or more than 1 year for patients to recover. There are no complementary treatments, beyond immunotherapy, to accelerate this recovery. Previous studies showed that SGE-301, a synthetic analogue of 24(S)-hydroxycholesterol, which is a potent and selective positive allosteric modulator of NMDAR, reverted the memory deficit caused by phencyclidine (a non-competitive antagonist of NMDAR), and prevented the NMDAR dysfunction caused by patients' NMDAR antibodies in cultured neurons. An advantage of SGE-301 is that it is optimized for systemic delivery such that plasma and brain exposures are sufficient to modulate NMDAR activity. Here, we used SGE-301 to confirm that in cultured neurons it prevented the antibody-mediated reduction of receptors, and then we applied it to a previously reported mouse model of passive cerebroventricular transfer of patient's CSF antibodies. Four groups were established: mice receiving continuous (14-day) infusion of patients' or controls' CSF, treated with daily subcutaneous administration of SGE-301 or vehicle (no drug). The effects on memory were examined with the novel object location test at different time points, and the effects on synaptic levels of NMDAR (assessed with confocal microscopy) and plasticity (long-term potentiation) were examined in the hippocampus on Day 18, which in this model corresponds to the last day of maximal clinical and synaptic alterations. As expected, mice infused with patient's CSF antibodies, but not those infused with controls' CSF, and treated with vehicle developed severe memory deficit without locomotor alteration, accompanied by a decrease of NMDAR clusters and impairment of long-term potentiation. All antibody-mediated pathogenic effects (memory, synaptic NMDAR, long-term potentiation) were prevented in the animals treated with SGE-301, despite this compound not antagonizing antibody binding. Additional investigations on the potential mechanisms related to these SGE-301 effects showed that (i) in cultured neurons SGE-301 prolonged the decay time of NMDAR-dependent spontaneous excitatory postsynaptic currents suggesting a prolonged open time of the channel; and (ii) it significantly decreased, without fully preventing, the internalization of antibody-bound receptors suggesting that additional, yet unclear mechanisms, contribute in keeping unchanged the surface NMDAR density. Overall, these findings suggest that SGE-301, or similar NMDAR modulators, could potentially serve as complementary treatment for anti-NMDAR encephalitis and deserve future investigations. [ABSTRACT FROM AUTHOR]

As 2020 is "The Year of the Rat" in the Chinese astrological calendar, it seems an appropriate time to consider whether we should bring back the laboratory rat to front-and-center in research on the basic biology of mammalian aging. Beginning in the 1970s, aging research with rats became common, peaking in 1992 but then declined dramatically by 2018 as the mouse became preeminent. The purpose of this review is to highlight some of the historical contributions as well as current advantages of the rat as a mammalian model of human aging, because we suspect at least a generation of researchers is no longer aware of this history or these advantages. Herein, we compare and contrast the mouse and rat in the context of several biological domains relevant to their use as appropriate models of aging: phylogeny/domestication, longevity interventions, pathology/physiology, and behavior/cognition. It is not the goal of this review to give a complete characterization of the differences between mice and rats, but to provide important examples of why using rats as well as mice is important to advance our understanding of the biology of aging. [ABSTRACT FROM AUTHOR]

The primary structure of canonical amyloid-β-protein was elucidated more than 30 years ago, yet the forms of amyloid-β that play a role in Alzheimer's disease pathogenesis remain poorly defined. Studies of Alzheimer's disease brain extracts suggest that amyloid-β, which migrates on sodium dodecyl sulphate polyacrylamide gel electrophoresis with a molecular weight of ∼7 kDa (7kDa-Aβ), is particularly toxic; however, the nature of this species has been controversial. Using sophisticated mass spectrometry and sensitive assays of disease-relevant toxicity we show that brain-derived bioactive 7kDa-Aβ contains a heterogeneous mixture of covalently cross-linked dimers in the absence of any other detectable proteins. The identification of amyloid-β dimers may open a new phase of Alzheimer's research and allow a better understanding of Alzheimer's disease, and how to monitor and treat this devastating disorder. Future studies investigating the bioactivity of individual dimers cross-linked at known sites will be critical to this effort. [ABSTRACT FROM AUTHOR]

Conserved noncoding elements (CNEs) have a significant regulatory influence on their neighboring genes. Loss of proximity to CNEs through genomic rearrangements can, therefore, impact the transcriptional states of the cognate genes. Yet, the evolutionary implications of such chromosomal alterations have not been studied. Through genome-wide analysis of CNEs and the cognate genes of representative species from five different mammalian orders, we observed a significant loss of genes' linear proximity to CNEs in the rat lineage. The CNEs and the genes losing proximity had a significant association with fetal, but not postnatal, brain development as assessed through ontology terms, developmental gene expression, chromatin marks, and genetic mutations. The loss of proximity to CNEs correlated with the independent evolutionary loss of fetus-specific upregulation of nearby genes in the rat brain. DNA breakpoints implicated in brain abnormalities of germline origin had significant representation between a CNE and the gene that exhibited loss of proximity, signifying the underlying developmental tolerance of genomic rearrangements that allowed the evolutionary splits of CNEs and the cognate genes in the rodent lineage. Our observations highlighted a nontrivial impact of chromosomal rearrangements in shaping the evolutionary dynamics of mammalian brain development and might explain the loss of brain traits, like cerebral folding of the cortex, in the rodent lineage. [ABSTRACT FROM AUTHOR]

Age-related changes in primary lymphoid organs are well described. Less is known about age-related changes affecting peripheral lymphoid organs, although defects in old peripheral lymph nodes (pLNs) were recently described in both steady state and during viral infection. To address whether such pLN defects were intrinsic to old T cells or extrinsic (due to aging microenvironment), we employed heterochronic parabiosis. We found no age-related intrinsic or extrinsic barriers to T cell circulation and seeding of pLN, spleen, and bone marrow. However, heterochronic parabiosis failed to improve cellularity of old pLN, suggesting an environment-based limit on pLN cellularity. Furthermore, upon parabiosis, pLN of the adult partner exhibited reduced, old-like stromal and T cell cellularity, which was restored following separation of parabionts. Decay measurement of adult and old T cell subsets following separation of heterochronic parabionts delineated both T cell-intrinsic and environmental changes in T cell maintenance. Moreover, parabiotic separation revealed differences between CD4 and CD8 T cell subset maintenance with aging, the basis of which will require further investigation. Reasons for this asymmetric and subset-specific pattern of differential maintenance are discussed in light of possible age-related changes in lymph nodes as the key sites for peripheral T cell maintenance. [ABSTRACT FROM AUTHOR]

Background: In the current study, we determined the effects of interleukin (IL)-21 on human natural killer (NK) cells and monocyte responses during Mycobacterium tuberculosis (Mtb) infection.Methods: We found that Mtb stimulated CD4+ and NK T cells from healthy individuals with latent tuberculosis infection (LTBI+) are major sources of IL-21. CD4+ cells from tuberculosis patients secreted less IL-21 than did CD4+ cells from healthy LTBI+ individuals. Interleukin-21 had no direct effect on Mtb-stimulated monocytes.Results: Interleukin-21-activated NK cells produced interferon (IFN)-γ, perforin, granzyme B, and granulysin; lysed Mtb-infected monocytes; and reduced Mtb growth. Interleukin-21-activated NK cells also enhanced IL-1β, IL-18, and CCL4/macrophage-inflammatory protein (MIP)-1β production and reduced IL-10 production by Mtb-stimulated monocytes. Recombinant IL-21 (1) inhibited Mtb growth, (2) enhanced IFN-γ, IL-1β, IL-18, and MIP-1β, and (3) reduced IL-10 expression in the lungs of Mtb-infected Rag2 knockout mice.Conclusions: These findings suggest that activated T cells enhance NK cell responses to lyse Mtb-infected human monocytes and restrict Mtb growth in monocytes through IL-21 production. Interleukin-21-activated NK cells also enhance the immune response by augmenting IL-1β, IL-18, and MIP-1β production and reducing IL-10 production by monocytes in response to an intracellular pathogen. [ABSTRACT FROM AUTHOR]

Nutritional ketosis is induced by high fat/low carbohydrate dietary regimens, which produce high levels of circulating ketone bodies, shifting metabolism away from glucose utilization. While ketogenic diets (KD) were initially introduced to suppress seizures, they are garnering attention for their potential to treat a myriad of neurodegenerative and metabolic disorders that are associated with advanced age. The feasibility and physiological impact of implementing a long-term KD in old animals, however, has not been systematically examined. In this study, young and aged rats consumed a calorically- and nutritionally-matched KD or control diet for 12 weeks. All KD-fed rats maintained higher levels of BHB and lower levels of glucose relative to controls. However, it took the aged rats longer to reach asymptotic levels of BHB compared to young animals. Moreover, KD-fed rats had significantly less visceral white and brown adipose tissue than controls without a loss of lean mass. Interestingly, the KD led to significant alterations in protein levels of hippocampal transporters for monocarboxylates, glucose, and vesicular glutamate and gamma-aminobutyric acid. Most notably, the age-related decline in vesicular glutamate transporter expression was reversed by the KD. These data demonstrate the feasibility and potential benefits of KDs for treating age-associated neural dysfunction. [ABSTRACT FROM AUTHOR]

Background: Standard cardiopulmonary resuscitation (CPR) is ineffective in treating traumatic cardiac arrest (TCA) following hemorrhagic shock despite fluid resuscitation. CPR adjuncts, including abdominal compressions and external counter pressure, have shown some success in laboratory settings. The Abdominal Aortic and Junctional Tourniquet (AAJT) is a device that occludes both venous and arterial blood at the level of the aortic bifurcation and likely increases thoracic pressure when applied to the abdomen. We developed a swine model of controlled hemorrhage to induce a state of TCA to test the ability of the AAJT to improve the efficacy of CPR.Methods: Twelve splenectomized, Yorkshire, male swine (70-90 kg) were randomized into two groups: presence or absence of AAJT placement. Controlled hemorrhage was performed at a rate of 2 mL/kg/min until systolic blood pressure reached below 10 mm Hg (defined as cardiac arrest). Following 3 minutes of arrest, the animals underwent CPR using a mechanical compression device along with either the presence or absence of the AAJT. Concurrently, 5 units of whole blood (2,500 mL) were delivered through the jugular vein at 500 mL/min. Efficacy of CPR was assessed by analyzing rates of return of spontaneous circulation (ROSC) and survival. Blood pressure, carotid blood flow, and other hemodynamic values were also compared.Findings: No significant differences between groups were observed before treatments. The controlled hemorrhage resulted in an average loss of 2,654 ± 323 g of blood over 18.2 ± 3.9 minutes. All animals that had a ROSC survived to the end of the 1-hour observation period. Animals with AAJT survived 83% (5/6) compared to 17% (1/6) of animals without AAJT. Finally, blood pressure, carotid flow, mean pulmonary artery pressure, and end tidal carbon dioxide were all significantly different between groups at the end of the first 10-minute compression period.Discussion/impact/recommendations: These results suggest that the AAJT could allow for increased CPR efficacy in cases of TCA when used in conjunction with rapid, massive blood transfusions. [ABSTRACT FROM AUTHOR]

Background. Chronic inflammatory and peripheral neuropathic pain (PNP) is a major health problem for which effective drug treatment is lacking. The pathophysiology of these debilitating conditions is incompletely understood, but nerve growth factor (NGF) is believed to play a major role. NGFantagonism has previously been shown to prevent pain hypersensitivity in rodent models of acute inflammatory pain and PNP, but most of those animal studies did not address the more clinically relevant issue of whether NGF-antagonism provides relief of established chronic pain behavior. Therefore, the aim of this study was to investigate whether blocking NGF actions with a humanized anti-NGF monoclonal antibody (PG110) would reverse/ attenuate established pain hypersensitivity in rat models of chronic/persistent inflammatory pain and PNP. Methods. The complete Freund's adjuvant (CFA) rat model of persistent inflammatory pain, and the L5 spinal nerve axotomy (SNA) model of PNP, were used in the present study. The effect of a single intravenous injection (10, 30, and 300 μg/kg) of an anti-NGF antibody PG110 on heat and mechanical hypersensitivity was assessed 5 and 7 days after CFA and SNA, respectively. Results. Compared to vehicle treated group, PG110 dose dependently attenuated established heat and mechanical hypersensitivity induced by CFA, but not that induced by SNA. The anti-allodynic and anti-hyperalgesic effects of PG110 in the CFA model were similar to those of the positive control naproxen (30mg/kg, i.v.). Conclusion. These findings suggest that therapies that target NGF or its receptors may be effective for treatment of persistent/chronic inflammatory pain, but probably not PNP. [ABSTRACT FROM AUTHOR]

Study Question: In comparison to in vivo development, how do different conditions of in vitro culture ('one step' versus 'sequential medium') impact DNA methylation and hydroxymethylation in preimplantation embryos?Summary Answer: Using rabbit as a model, we show that DNA methylation and hydroxymethylation are both affected by in vitro culture of preimplantation embryos and the effect observed depends on the culture medium used.What Is Known Already: Correct regulation of DNA methylation is essential for embryonic development and DNA hydroxymethylation appears more and more to be a key player. Modifications of the environment of early embryos are known to have long term effects on adult phenotypes and health; these probably rely on epigenetic alterations.Study Design Size, Duration: The study design we used is both cross sectional (control versus treatment) and longitudinal (time-course). Each individual in vivo experiment used embryos flushed from the donor at the 2-, 4-, 8-, 16- or morula stage. Each stage was analyzed in at least two independent experiments. Each individual in vitro experiment used embryos flushed from donors at the 1-cell stage (19 h post-coïtum) which were then cultured in parallel in the two tested media until the 2-, 4-, 8- 16-cell or morula stages. Each stage was analyzed in at least three independent experiments. In both the in vivo and in vitro experiments, 4-cell stage embryos were always included as an internal reference.Participants/materials, Setting, Methods: Immunofluorescence with antibodies specific for 5-methylcytosine (5meC) and 5-hydroxymethylcytosine (5hmeC) was used to quantify DNA methylation and hydroxymethylation levels in preimplantation embryos. We assessed the expression of DNA methyltransferases (DNMT), of ten eleven translocation (TET) dioxigenases and of two endogenous retroviral sequences (ERV) using RT-qPCR, since the expression of endogenous retroviral sequences is known to be regulated by DNA methylation. Three repeats were first done for all stages; then three additional repetitions were performed for those stages showing differences or tendencies toward differences between the different conditions in the first round of quantification.Main Results and the Role Of Chance: The kinetics of DNA methylation and hydroxymethylation were modified in in vitro cultured embryos, and the observed differences depended on the type of medium used. These differences were statistically significant. In addition, the expression of TET1 and TET2 was significantly reduced in post-embryonic genome activation (EGA) embryos after in vitro culture in both tested conditions. Finally, the expression of two retroviral sequences was analyzed and found to be significantly affected by in vitro culture.Limitations Reasons For Caution: Our study remains mostly descriptive as no direct link can be established between the epigenetic changes observed and the expression changes in both effectors and targets of the studied epigenetic modifications. The results we obtained suggest that gene expression could be affected on a large scale, but this remains to be confirmed.Wider Implications Of the Findings: Our results are in agreement with the literature, showing that DNA methylation is sensitive to in vitro culture. As we observed an effect of both tested culture conditions on the tested epigenetic marks and on gene expression, we cannot conclude which medium is potentially closest to in vivo conditions. However, as the observed effects are different, additional studies may provide more information and potential recommendations for the use of culture media in assisted reproductive technology.Study Funding/competing Interests: This work was supported by an 'AMP diagnostic prénatal et diagnostic génétique' 2012 grant from the French Agence de la Biomédecine. This study was performed within the framework of ANR LABEX 'REVIVE' (ANR-10-LABX-73). Authors are members of RGB-Net (TD 1101) and Epiconcept (FA 1201) COST actions. The authors declare that there is no competing interest. [ABSTRACT FROM AUTHOR]

With aging, there is a decline in cardiac function accompanying increasing risk of arrhythmias. These effects are likely to be mechanistically associated with age-associated changes in calcium regulation within cardiac myocytes. Previous studies suggest that lifelong exercise can potentially reduce age-associated changes in the heart. Although exercise itself is associated with changes in cardiac function, little is known about the interactions of aging and exercise with respect to myocyte calcium regulation. To investigate this, adult (12 months) and old (24 months) C57/Bl6 mice were trained using moderate-intensity treadmill running. In response to 10 weeks' training, comparable cardiac hypertrophic responses were observed, although aging independently associated with additional cardiac hypertrophy. Old animals also showed increased L- and T-type calcium channels, the sodium-calcium exchange, sarcoendoplasmic reticulum calcium ATPase, and collagen (by 50%, 92%, 66%, 88%, and 113% respectively). Short-term exercise training increased D-type and T-type calcium channels in old animals only, whereas an increase in sodium-calcium exchange was seen only in adult animals. Long-term (12 months) training generally opposed the effects of aging. Significant hypertrophy remained in long-term trained old animals, but levels of sarcoendoplasmic reticulum calcium ATPase, sodium-calcium exchange, and collagen were not significantly different from those found in the adult trained animals. [ABSTRACT FROM AUTHOR]

Objectives: Biofilm-associated implant infections represent a serious public health problem. Covalent immobilization of antimicrobial agents on titanium (Ti), thereby inhibiting biofilm formation of microbial pathogens, is a solution to this problem.Methods: Vancomycin (VAN) and caspofungin (CAS) were covalently bound on Ti substrates using an improved processing technique adapted to large-scale coating of implants. Resistance of the VAN-coated Ti (VAN-Ti) and CAS-coated Ti (CAS-Ti) substrates against in vitro biofilm formation of the bacterium Staphylococcus aureus and the fungal pathogen Candida albicans was determined by plate counting and visualized by confocal laser scanning microscopy. The efficacy of the coated Ti substrates was also tested in vivo using an adapted biomaterial-associated murine infection model in which control-Ti, VAN-Ti or CAS-Ti substrates were implanted subcutaneously and subsequently challenged with the respective pathogens. The osseointegration potential of VAN-Ti and CAS-Ti was examined in vitro using human bone marrow-derived stromal cells, and for VAN-Ti also in a rat osseointegration model.Results: In vitro biofilm formation of S. aureus and C. albicans on VAN-Ti and CAS-Ti substrates, respectively, was significantly reduced compared with biofilm formation on control-Ti. In vivo, we observed over 99.9% reduction in biofilm formation of S. aureus on VAN-Ti substrates and 89% reduction in biofilm formation of C. albicans on CAS-Ti substrates, compared with control-Ti substrates. The coated substrates supported osseointegration in vitro and in vivo.Conclusions: These data demonstrate the clinical potential of covalently bound VAN and CAS on Ti to reduce microbial biofilm formation without jeopardizing osseointegration. [ABSTRACT FROM AUTHOR]

Background: Resistance to the neuraminidase inhibitor oseltamivir can be conferred by a well-characterized mutation in the neuraminidase gene, H275Y. In human H1N1 viruses that circulated in the first years of the 21st century, this mutation carried a fitness cost and resistant viruses were rare. During the 2007-08 influenza season, oseltamivir-resistant viruses of H1N1 phenotype emerged and predominated. March 2009 saw the emergence of a novel H1N1 influenza pandemic. We examined whether the H275Y mutation affected neuraminidase enzyme activity or replication of the pandemic influenza virus.Methods: Using reverse genetics we engineered the H275Y mutation into the neuraminidase of a 2009 pandemic H1N1 virus and assessed the ability of this enzyme to desialylate mono- and multivalent substrates. The growth kinetics of wild-type and mutant viruses were assessed in Madin-Darby canine kidney (MDCK) and fully differentiated human airway epithelial (HAE) cells.Results: The presence of H275Y was associated with a 1.3-fold decrease in the affinity of the neuraminidase for a monovalent substrate and a 4-fold compromise in desialylation of multivalent substrate. This was associated with a fitness cost to viral replication in vitro, which only became apparent during competitive replication in the mucus-rich HAE culture system.Conclusions: The neuraminidase protein of pandemic influenza isolates tolerates the H275Y mutation and this mutation confers resistance to oseltamivir. However, unlike seasonal H1N1 viruses isolated since 2007, the mutation is not associated with any fitness advantage and thus is unlikely to predominate without further antigenic drift, compensating mutations or intense selection pressure. [ABSTRACT FROM AUTHOR]

Background: ATP-sensitive K(+) (K(ATP)) channels link intracellular metabolism with membrane excitability and play crucial roles in cellular physiology and protection. The K(ATP) channel protein complex is composed of pore forming, Kir6.x (Kir6.1 or Kir6.2) and regulatory, SURx (SUR2A, SUR2B or SUR1), subunits that associate in different combinations. The objective of this study was to determine whether mammalian oocytes (human, bovine, porcine) express K(ATP) channels.Methods: Supernumerary human oocytes at different stages of maturation were obtained from patients undergoing assisted conception treatments. Bovine and porcine oocytes in the germinal vesicle (GV) stage were obtained by aspirating antral follicles from abattoir-derived ovaries. The presence of mRNA for K(ATP) channel subunits was determined using real-time RT-PCR with primers specific for Kir6.2, Kir6.1, SUR1, SUR2A and SUR2B. To assess whether functional K(ATP) channels are present in human oocytes, traditional and perforated patch whole cell electrophysiology and immunoprecipitation/western blotting were used.Results: Real-time PCR revealed that mRNA for Kir6.1, Kir6.2, SUR2A and SUR2B, but not SUR1, were present in human oocytes of different stages. Only SUR2B and Kir6.2 mRNAs were detected in GV stage bovine and porcine oocytes. Immunoprecipitation with SUR2 antibody and western blotting with Kir6.1 antibody identified bands corresponding to these subunits in human oocytes. In human oocytes, 2,4-dinitrophenol (400 µM), a metabolic inhibitor known to decrease intracellular ATP and activate K(ATP) channels, increased whole cell K(+) current. On the other hand, K(+) current induced by low intracellular ATP was inhibited by extracellular glibenclamide (30 µM), an oral antidiabetic known to block the opening of K(ATP) channels.Conclusions: In conclusion, mammalian oocytes express K(ATP) channels. This opens a new avenue of research into the complex relationship between metabolism and membrane excitability in oocytes under different conditions, including conception. [ABSTRACT FROM AUTHOR]

Objectives: Expression of fungal virulence factors can be influenced by exposure to antifungal agents. To test this hypothesis, we determined the effects of subinhibitory concentrations of three antifungal agents on expression of three secreted proteinase genes associated with virulence in filamentous forms of Candida albicans.Methods: GFP-SAP promoter constructs and fluorescence measurement, transcript profiling and RT-PCR in vitro and in an animal model of disseminated Candida infection.Results: Exposure of C. albicans to subinhibitory concentrations of fluconazole in RPMI 1640 in the absence of serum led to up-regulation of the virulence-associated genes SAP4, SAP5 and SAP6 in hyphae and long pseudohyphae. Measurements with green fluorescent protein (GFP)-tagged promoters showed that the fluorescence of SAP4 and SAP6 under these conditions was strongest in the apical tip compartments of these filamentous cells and declined in compartments more proximal to the parent yeast cell. By contrast, SAP5-GFP fluorescence was expressed at similar levels in all cell compartments. Exposure to fluconazole led to significant increases in GFP-SAP4 and -SAP6 fluorescence in the filaments; itraconazole exposure also significantly increased GFP-SAP4 fluorescence, whereas flucytosine had no effect on any of the constructs. In experimentally infected animals, fluorescence of the GFP-SAP promoter fungal cells in kidney tissues was greater than that was seen in vitro for all four SAP constructs: treatment of animals with fluconazole did not significantly increase SAP promoter expression as measured by GFP fluorescence.Conclusions: Azole antifungal agents stimulated up-regulation of SAP4 and SAP6 genes in filamentous C. albicans cells in vitro and may therefore influence virulence as well as growth of the fungus. However, such effects appear to be transient in vivo. [ABSTRACT FROM AUTHOR]

How growth hormone (GH) stimulates protein synthesis is unknown. Phosphoinositide 3-kinase/Akt/mammalian target of rapamycin (PI3K/Akt/mTOR) signaling pathways balance anabolic and catabolic activities in response to nutrients and growth factor signaling. As a test of GH signaling, immunoassays of two downstream translation regulatory proteins were compared in ad libitum-fed 2-month-old normal and Ames (Prop1df) dwarf mice. Phosphorylation of the p70 and p85 isoforms of S6 kinase 1 in liver and the p70 isoform in gastrocnemius muscle were significantly decreased in dwarfs. Messenger RNA (mRNA) Cap-binding demonstrated significantly higher levels of translation repressor 4E-BP1/eukaryotic initiation factor 4E (eIF4E) (coprecipitates) from dwarf livers, but not muscle. Consistent with these binding data, significantly less phosphorylation of 4E-BP1 was documented in dwarf liver. These data suggest a link between GH signaling and translation control in a model of extended longevity. [ABSTRACT FROM AUTHOR]

We analyzed the effect of oral melatonin (23 microg/0.1 ml/animal/d; 1 h before dark on 12 consecutive days) in old birds, in natural photoperiods, on the hormone's plasma levels, and phagocytosis. Blood collections were performed daily at 2:00 am and 4:00 pm until 5 days after the treatment. From day 1, the melatonin levels were significantly higher than basal levels at both times. Values at 2:00 am were significantly higher than the 4:00 pm values. After treatment, the melatonin levels declined, returning from day 14 to basal values at both hours. At 2:00 am, phagocytosis was significantly greater than that obtained at 4:00 pm and greater than basal values. The 4:00 pm values were only significantly greater than basal on days 6 and 8, parallel to a decline in superoxide anion levels, which were lowest at 2:00 am. Melatonin administered to old ring doves increases the differences between nocturnal and diurnal plasma levels, and, in parallel, increases phagocytosis and reduces superoxide radical levels in heterophils. [ABSTRACT FROM AUTHOR]

We examined the role of heme oxygenase (HO)-1 in morphine-induced decrease in macrophage migration. Morphine promoted expression of HO-1 in murine macrophages. Morphine-receiving mice (MRCs) showed decreased (P<.001) macrophage migration and increased (P<.001) occurrence of macrophage apoptosis. In in vitro studies, peritoneal macrophages harvested from MRCs also showed decreased (P<.001) migration, compared with those from control mice. Bone marrow cells isolated from MRCs showed not only decreased (P<.001) migration but also increased apoptosis. Pretreatment of MRCs with hemin not only decreased migration of macrophages further but also enhanced the apoptosis of peritoneal macrophages. On the other hand, pretreatment of MRCs with zinc protoporphyrin attenuated the effect of morphine on both macrophage migration and the occurrence of apoptosis. In in vitro studies, pretreatment of macrophages with hemin exacerbated morphine-induced apoptosis, whereas pretreatment with zinc protoporphyrin attenuated morphine-induced macrophage apoptosis. [ABSTRACT FROM AUTHOR]

Background: The expression level of several matrix metalloproteinases (MMPs), including MMP-2 and MMP-9, in ovarian cancer cells is directly associated with their invasive and metastatic potentials. MMP-9 is also expressed in stromal cells adjacent to the tumor. To investigate the contribution of MMP-9 expression in stromal cells to ovarian tumor growth, we examined angiogenesis and progressive growth of human ovarian cancer cells implanted into mice with and without the MMP-9 gene.Methods: Human ovarian cancer cells SKOV3.ip1 and HEY-A8 were implanted into the peritoneal cavities of nude mice that lacked the gene for MMP-9 (MMP-9(-/-)) or were wild type for MMP-9 (MMP-9(+/+)) (10 mice of each genotype per cell line). Tumor incidence, tumor size, and volume of ascites fluid were recorded for each mouse at 30 and 45 days after HEY-A8 and SKOV3.ip1 cell injections, respectively. Blood vessel density and macrophage infiltration into the lesions were analyzed in excised tumors by immunohistochemistry and double immunofluorescence. Tumor growth was also studied in MMP-9(-/-) nude mice that had been reconstituted with spleen cells collected from either MMP-9(+/+) or MMP-9(-/-) nude mice. All statistical tests were two-sided.Results: HEY-A8 cells expressed high levels of MMP-9, and SKOV3.ip1 cells expressed low levels. Nevertheless, tumor incidence and growth were statistically significantly lower in MMP-9(-/-) mice than in MMP-9(+/+) mice injected with cells from either line (for tumor size, P =.006 and.042 for HEY-A8 and SKOV3.ip1 cells, respectively). Compared with MMP-9(+/+) mice injected with human ovarian cancer cells, MMP-9(-/-) mice injected with human ovarian cancer cells displayed decreased microvessel density and decreased macrophage infiltration into the lesions. Compared with MMP-9(-/-) mice that received spleen cells (a rich source of macrophages) from MMP-9(-/-) mice, those that received spleen cells from MMP-9(+/+) mice before cancer cell injections displayed increased angiogenesis and tumorigenicity of the cancer cells. The growing tumors contained MMP-9-expressing macrophages.Conclusion: Host-derived MMP-9 expression, most likely in tumor-infiltrating macrophages, appears to play a critical role in angiogenesis and progressive growth of human ovarian tumors in mice. [ABSTRACT FROM AUTHOR]

In this study, we examined the effects of age and growth hormone replacement on both coronary blood flow and capillary density. Blood flow was measured by using [(14)C]-iodoantipyrine in three groups of anesthetized Brown Norway x Fischer 344 rats: young vehicle-treated animals (6 months; n = 13), old vehicle treated animals (30 months; n = 9), and old animals treated with bovine growth hormone (200 microg/kg) twice a day for 30 days (30 months; n = 7). Capillary density was measured by color segmentation analysis of sections stained for platelet endothelial cell adhesion molecule-1. In all regions examined, coronary blood flow decreased with age, and growth hormone administration resulted in an increase in flow compared to vehicle-treated animals. Capillary density decreased with age in the apex and the left ventricular middle segment. In response to growth hormone administration, capillary density increased significantly in the apex but not in other regions of the heart. Our results demonstrate that growth hormone enhances regional myocardial blood flow in the aged heart and suggest that part of this effect could be due to an increase in capillary density. [ABSTRACT FROM AUTHOR]

Impaired reinnervation has been implicated as the cause of the threefold disparity in the recovery of maximum force (P0) of standard muscle grafts in old compared with young rats. The specific, null hypothesis of this study is that compared with age-matched control extensor digitorum longus (EDL) muscles, nerve-intact EDL muscle grafts in young and old rats show no evidence of an age-related impairment in reinnervation. Nerve-intact grafts were performed in 3-month-old and 23-month-old rats and were evaluated 60 days postoperatively. Compared with age-matched control EDL muscles, nerve-intact grafts in young and old rats showed no difference in muscle mass or motor unit numbers. The mean motor unit P0 for nerve-intact graft muscles in both age groups was significantly lower than that of age-matched control muscles. These data support our hypothesis that if axons are allowed to regenerate in an endoneurial environment, there is no evidence of an age-related impairment in muscle reinnervation. [ABSTRACT FROM AUTHOR]

The purpose of this study was to determine if masseter muscle endurance changes with increasing age and, if so, to examine mechanisms of fatigue. Characteristics of fatigue were measured under isometric conditions using high-frequency stimulation of anterior deep masseter (ADM) muscles of male Fischer 344 rats, 5 to 24 months old, and fed a hard (HD) or a soft (SD) diet. Potentiating effects of caffeine on ADM muscle performance in vitro were also examined. Fatigability increased by 48% with age in muscles of HD rats. Muscles of SD rats were highly fatigable at all ages. Increased HD fatigability was associated with significantly decreased concentrations of Na+/K+-adenosine triphosphatase (22%) and decreased responsiveness to caffeine postfatigue (29%). The pH levels decreased similarly in fatigued muscles of all groups. We conclude that the age-related increase in fatigability is associated with alterations in excitation-contraction coupling mechanisms. However, differences between SD and HD on ADM muscles represent possible fiber-type transitions. [ABSTRACT FROM AUTHOR]

Background and Methods: Breast cancer resistance protein (BCRP/MXR/ABCP) is a multidrug-resistance protein that is a member of the adenosine triphosphate-binding cassette family of drug transporters. BCRP can render tumor cells resistant to the anticancer drugs topotecan, mitoxantrone, doxorubicin, and daunorubicin. To investigate the physiologic role of BCRP, we used polarized mammalian cell lines to determine the direction of BCRP drug transport. We also used the BCRP inhibitor GF120918 to assess the role of BCRP in protecting mice against xenobiotic drugs. Bcrp1, the murine homologue of BCRP, was expressed in the polarized mammalian cell lines LLC-PK1 and MDCK-II, and the direction of Bcrp1-mediated transport of topotecan and mitoxantrone was determined. To avoid the confounding drug transport provided by P-glycoprotein (P-gp), the roles of Bcrp1 in the bioavailability of topotecan and the effect of GF120918 were studied in both wild-type and P-gp-deficient mice and their fetuses.Results: Bcrp1 mediated apically directed transport of drugs in polarized cell lines. When both topotecan and GF120918 were administered orally, the bioavailability (i.e., the extent to which a drug becomes available to a target tissue after administration) of topotecan in plasma was dramatically increased in P-gp-deficient mice (greater than sixfold) and wild-type mice (greater than ninefold), compared with the control (i.e., vehicle-treated) mice. Furthermore, treatment with GF120918 decreased plasma clearance and hepatobiliary excretion of topotecan and increased (re-)uptake by the small intestine. In pregnant GF120918-treated, P-gp-deficient mice, relative fetal penetration of topotecan was twofold higher than that in pregnant vehicle-treated mice, suggesting a function for BCRP in the maternal-fetal barrier of the placenta.Conclusions: Bcrp1 mediates apically directed drug transport, appears to reduce drug bioavailability, and protects fetuses against drugs. We propose that strategic application of BCRP inhibitors may thus lead to more effective oral chemotherapy with topotecan or other BCRP substrate drugs. [ABSTRACT FROM AUTHOR]

Caloric restriction (CR), which increases longevity and retards age-associated diseases in laboratory rodents, is being evaluated in nonhuman primate trials. CR reduces oxidative stress in rodents and appears to improve risk factors for cardiovascular disease in nonhuman primates. We tested the hypothesis that low-density lipoprotein (LDL) oxidizability is reduced in two monkey species (rhesus and cynomolgus) subjected to chronic CR. In both species, no significant differences occurred between CR and control animals on total, LDL, or high-density lipoprotein (HDL) cholesterol. In rhesus monkeys, triglycerides were higher in controls than CR (139 +/- 23 vs 66 +/- 8 mg/dl,p < .01, respectively). LDL from CR rhesus monkeys was reduced in triglyceride content and molecular weight compared to controls, whereas LDL composition in cynomolgus monkeys was similar in CR and control animals. In keeping with minor deviations in lipids, antioxidants, and LDL composition, no consistent differences in in vitro LDL oxidizability were apparent between CR and controls in either species. [ABSTRACT FROM AUTHOR]

Despite the fact that the anxiety-relieving effects of smoking are widely reported by smokers, human laboratory studies have not found consistent evidence for this effect. In animals, contrasting results also have been reported, with Sprague-Dawley rats unaffected by nicotine in behavioral tests of anxiety but with other rat strains (e.g., Wistar, Fischer-344) displaying behaviors indicative of anxiolysis. The present experiment used the social interaction test to evaluate effects of nicotine (12 mg/kg/day) chronically administered via minipump on social and non-social behaviors in 48 male and 48 female Long-Evans rats--a strain that has not previously been evaluated and that is known to differ from Sprague-Dawleys in other behavioral responses to nicotine. Because environmental conditions can alter behavioral effects of drugs, animals lived in either individual or same-sex group housing. Social interactions were measured on day 10 of drug administration and social behaviors (touch, follow, sniff-other, wrestle) as well as non-social behaviors (anxiety-related behaviors: freezing, grooming-self; exploratory behavior: moving) were scored. Group housing decreased social behaviors and increased anxiety-related behaviors. Nicotine's effects depended upon housing condition, such that nicotine further decreased social behaviors and increased anxiety-related behaviors of group-housed animals, with weaker effects in single-housed animals. Overall, the results suggest that nicotine's effects are modified by environmental conditions, and that nicotine administration at this dosage and in this particular rat strain results in anxiogenic effects. [ABSTRACT FROM AUTHOR]

The complexity of tobacco smoke leads to some confusion about the mechanisms by which it causes lung cancer. Among the multiple components of tobacco smoke, 20 carcinogens convincingly cause lung tumors in laboratory animals or humans and are, therefore, likely to be involved in lung cancer induction. Of these, polycyclic aromatic hydrocarbons and the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone are likely to play major roles. This review focuses on carcinogens in tobacco smoke as a means of simplifying and clarifying the relevant information that provides a mechanistic framework linking nicotine addiction with lung cancer through exposure to such compounds. Included is a discussion of the mechanisms by which tobacco smoke carcinogens interact with DNA and cause genetic changes--mechanisms that are reasonably well understood--and the less well defined relationship between exposure to specific tobacco smoke carcinogens and mutations in oncogenes and tumor suppressor genes. Molecular epidemiologic studies of gene-carcinogen interactions and lung cancer--an approach that has not yet reached its full potential--are also discussed, as are inhalation studies of tobacco smoke in laboratory animals and the potential role of free radicals and oxidative damage in tobacco-associated carcinogenesis. By focusing in this review on several important carcinogens in tobacco smoke, the complexities in understanding tobacco-induced cancer can be reduced, and new approaches for lung cancer prevention can be envisioned. [ABSTRACT FROM AUTHOR]

We tested the hypothesis that after skeletal muscle regeneration in old compared with young rats damage to the motor nerve rather than damage to muscle fibers determines the magnitude of the deficits in muscle mass and maximum force (Po). The mass and Po of extensor digitorum longus (EDL) muscles of young (4 months) and old (24 months) male rats were compared two months following (i) Marcaine treatment plus simultaneous motor nerve transection, (ii) motor nerve transection alone, and (iii) Marcaine treatment alone (from data compiled previously). In both the nerve transection-only and Marcaine with nerve transection groups the recovery of mass and Po was significantly greater in young than in old rats. This is in contrast to our previous data showing that in the absence of nerve damage Marcaine-treated muscle in old rats regenerates as well as that in young rats. Our hypothesis was supported, and we conclude that impaired axonal regeneration, re-establishment of nerve-muscle contact, or both, is the critical component in the impaired regeneration of muscle grafts in old as compared with young rats. [ABSTRACT FROM AUTHOR]

Changes in carbohydrate expression on endometrial and blastocyst cell surfaces may play a critical role in the process of implantation. Le(y) is an oligosaccharide antigen which has been shown to be involved in blastocyst attachment in the mouse. In the present study, immunohistochemical distribution of Le(y) in endometrium during proliferative and secretory phases of normal menstrual cycles in the rhesus monkey was examined. Endometrial samples were collected on cycle days 7 (n=4), 13 (n= 4), 16 (n=4), 20 (n=4) and 25 (n=3). There was a gradual increase of Le(y) in luminal surface from proliferative to periovulatory (P < 0.001), and from periovulatory to postovulatory (P < 0.05), phases. Le(y) then remained constant in the midsecretory phase and decreased (P < 0.01) during the premenstrual phase. Le(y) score in glands did not change between the phases, except in midsecretory phase when it was higher than that in other phases (P < 0.05). The stromal compartment showed no statistically significant changes. The profiles of endometrial Le(y) on day 6 after ovulation in mated fecund cycles with or without early luteal phase mifepristone treatment were also examined. Females were allowed to cohabit with males during days 8-16 of their ovulatory cycles and were injected s.c. with vehicle (n=7) only, or with a single dose of mifepristone (2 mg/kg body weight; n=8) on day 2 after ovulation. Significant decreases in the area and optical absorption of immunoprecipitate were observed in the epithelial compartment (P < 0.01) following mifepristone treatment. There was no change in the stromal compartment either in area or in optical absorption of immunoprecipitate for Le(y) with or without mifepristone treatment. The expression of Le(y) in the endometrial epithelial compartment appears to be influenced by progesterone and may be associated with endometrial receptivity prior to implantation in the rhesus monkey. [ABSTRACT FROM AUTHOR]

Since alpha6beta1 integrin has been shown to function as a sperm adhesion receptor in the mouse, we investigated the potential role of beta1 integrin in the gamete fusion process in humans. The expression of beta1 integrin was morphologically analysed by indirect immunofluorescence and confocal microscopy. A homogeneous and intense staining was detected at the plasma membrane, and in some subcortical vesicles of germinal vesicle stage oocytes (GV). Beta1 almost disappeared from oolemma and cytoplasm of metaphase I (MI) oocytes, but was re-expressed as asymmetrical patches at the plasma membrane of metaphase II stage oocytes (MII). A functional fusion assay based on Hoechst or calcein-AM dye transfer from one gamete to the other showed that maturing oocytes were able to fuse with an increasing number of spermatozoa (11-22 from GV to MII respectively), and that fused spermatozoa co-localized with beta1 integrin patches. Human gamete fusion was only partially inhibited either by RGD-containing peptide (GRGDTP), or by blocking anti-human beta1 integrin monoclonal antibody (DE9), with a maximum of 50% inhibition. Despite the combined addition of GRGDTP and blocking mouse anti-human beta1 integrin DE9 in the assay, a complete inhibition of fusion could not be achieved. A mouse polyclonal antibody raised against human oocyte membranes was more potent in inhibiting the fusion. Since beta1 integrin expression at the plasma membrane was not correlated to oocyte fusibility, and since it was only partially inhibited by DE9 and/or RGD peptide, we suggest that human gamete fusion can bypass the beta1 requirement. Beta1 integrin certainly participates in human gamete fusion by acting in co-operation with multiple integrin/disintegrin couples or another cofactor, not yet identified. [ABSTRACT FROM AUTHOR]

The disassembly and reorganization of sperm-derived structures are landmarks for the onset of embryonic development. Since complete information on these events is not yet available, we examined the disassembly of the sperm axoneme, the formation of the sperm aster, and the decondensation and development of the male and female pronuclei in inseminated Rhesus monkey oocytes conceived by in-vitro fertilization (IVF) or by intracytoplasmic sperm injection. During IVF, the spermatozoa lose their acrosomes after contacting the zona pellucida, and the plasma membrane and nuclear envelope disappear after fusion with the oolemma. Subsequently, a sperm aster of microtubules forms around the proximal centriole, which is bound to the sperm connecting piece. This process is then followed by the formation of both pronuclei, which single sperm centriole later duplicates and the bipolar mitotic apparatus is observed. Following sperm injection, the spermatozoa have both an intact plasma membrane and acrosome. Although the microtubules form the sperm aster in a fashion identical to that seen during IVF, the presence of an intact acrosome appears to be associated with a heterogeneity in the decondensation of sperm chromatin. While this may indicate an abnormal pattern of chromatin decondensation during the formation of the male pronucleus following sperm injection, the male pronucleus eventually fully decondenses, as during IVF. Sperm mitochondria are displaced as the sperm centriole is exposed. Annulate lamellae and a previously undescribed organelle which seems to contain annulate lamellae precursors, as well as maternal mitochondria, are found in association with the developing pronuclear envelopes. This information increases understanding of fertilization in primates, and may also be of significance for use in assisted human reproduction as well as in the preservation of endangered mammalian species. In addition, these results demonstrates the similarities between fertilization in Rhesus monkeys and humans, providing additional evidence for the use of this non-human primate as a model system in which to investigate the cellular and molecular biological basis of human reproduction. [ABSTRACT FROM AUTHOR]