In Vitro Biofilm Activity of Non-Candida albicans Candida Species

Candidosis has been attributed to C. albicans; however, infections caused by non-Candida albicans Candida (NCAC) species are increasingly being recognised. The ability of Candida to grow as a biofilm is an important feature that promotes both infection and persistence in the host. The biofilms’ activity is significant since high activity might be associated with enhanced expression of putative virulence factors, whilst in contrast low activity has previously been suggested as a mechanism for resistance of biofilm cells to antimicrobials. The aim of this study was to determine the metabolic activity of in vitro biofilms formed by different clinical isolates of NCAC species. The in situ total metabolic activity of C. parapsilosis, C. tropicalis and C. glabrata biofilms was determined using 2,3-(2-methoxy-4-nitro-5-sulphophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide (XTT) reduction assay, and the number of cultivable cells was also established by CFU (colony forming unit) counts. The biofilm structure was assessed by scanning electron microscopy (SEM). Results showed that total biofilm metabolic activity was species and strain dependent. C. glabrata exhibited the lowest biofilm metabolic activity despite having the highest number of biofilm cultivable cells. Similarly, the metabolic activity of resuspended C. glabrata biofilm and planktonic cells was lower than that of the other species. This study demonstrates the existence of intrinsic activity differences amongst NCAC species, which could have important implications in terms of species relative virulence. Furthermore, the absence of an obvious correlation, between cultivable cells number and total biofilm activity, raises the question about which parameter is the most appropriate for the in vitro assessment of biofilms and their potential clinical significance.     

Rapid Estradiol-17β Modulation of Opioid Actions on the Electrical and Secretory Activity of Rat Oxytocin Neurons In vivo

During pregnancy, emergence of endogenous opioid inhibition of oxytocin neurons is revealed by increased oxytocin secretion after administration of the opioid receptor antagonist, naloxone. Here we show that prolonged estradiol-17β and progesterone treatment (mimicking pregnancy levels) potentiates naloxone-induced oxytocin secretion in urethane-anesthetized virgin female rats. We further show that estradiol-17β alone rapidly modifies opioid interactions with oxytocin neurons, by recording their firing rate in anesthetized rats sensitized to naloxone by morphine dependence. Naloxone-induced morphine withdrawal strongly increased the firing rate of oxytocin neurons in morphine dependent rats. Estradiol-17β did not alter basal oxytocin neuron firing rate over 30 min, but amplified naloxone-induced increases in firing rate. Firing pattern analysis indicated that acute estradiol-17β increased oxytocin secretion in dependent rats by increasing action potential clustering without an overall increase in firing rate. Hence, rapid estradiol-17β actions might underpin enhanced oxytocin neuron responses to naloxone in pregnancy. Special issue article in honor of George Fink.     

Activity-Dependent Downscaling of Subthreshold Synaptic Inputs during Slow-Wave-Sleep-like Activity In Vivo

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Neuroprotective Effect of Natural Products Against Alzheimer’s Disease

Nature has gifted mankind with a plethora of flora-bearing fruits, vegetables and nuts. The diverse array of bioactive nutrients present in these natural products plays a pivotal role in prevention and cure of various neurodegenerative diseases, such as Alzheimer's disease (AD), Parkinson's disease and other neuronal dysfunctions. Accumulated evidence suggests that naturally occurring phyto-compounds, such as polyphenolic antioxidants found in fruits, vegetables, herbs and nuts, may potentially hinder neurodegeneration, and improve memory and cognitive function. Nuts such as walnut have also demonstrated neuroprotective effect against AD. The molecular mechanisms behind the curative effects rely mainly on the action of phytonutrients on distinct signalling pathways associated with protein folding and neuroinflammation. The neuroprotective effects of various naturally occurring compounds in AD is evaluating in this review.     

In vitro and in vivo antioxidant activity of exopolysaccharide fractions from Bifidobacterium animalis RH

The aim of the study was to purify the exopolysaccharides (EPS) produced from Bifidobacterium animalis RH, which was isolated from the feces of Bama centenarians in Guangxi of China, and evaluate their antioxidant activities in vitro and in vivo. 2 fractions, a neutral EPS fraction (EPSa) and an acidic EPS fraction (EPSb), were obtained and compared for antioxidative activity. In vitro, they both showed remarkable inhibition effect on lipid peroxidation and strong DPPH radical scavenging activity, hydroxyl radical scavenging activity, superoxide radical scavenging activity, in which the last two were measured by the electron spin resonance (ESR). In vivo, EPSa and EPSb were orally administrated for 30 days in a d-galactose induced aged mice model. As results, they both could significantly increase the activities of SOD, CAT and total antioxidant capacity (TAOC) in serums and glutathione GST in livers. They also could inhibit significantly the formation of MDA in serums and livers, and reduce the activity of MAO and lipofuscin accumulation in mice brain. Moreover, EPSb exhibited much higher antioxidant activities than EPSa in vitro and in vivo. The results suggested that EPS fractions of Bifidobacterium animalis RH had direct and potent antioxidant activities.     

In vivo and in vitro function of human UDP-galactose 4'-epimerase variants

Type III galactosemia results from reduced activity of the enzyme UDP-galactose 4′-epimerase. Five disease-associated alleles (G90E, V94M, D103G, N34S and L183P) and three artificial alleles (Y105C, N268D, and M284K) were tested for their ability to alleviate galactose-induced growth arrest in a Saccharomyces cerevisiae strain which lacks endogenous UDP-galactose 4′-epimerase. For all of these alleles, except M284K, the ability to alleviate galactose sensitivity was correlated with the UDP-galactose 4′-epimerase activity detected in cell extracts. The M284K allele, however, was able to substantially alleviate galactose sensitivity, but demonstrated near-zero activity in cell extracts. Recombinant expression of the corresponding protein in Escherichia coli resulted in a protein with reduced enzymatic activity and reduced stability towards denaturants in vitro. This lack of stability may result from the introduction of an unpaired positive charge into a bundle of three α-helices near the surface of the protein. The disparities between the in vivo and in vitro data for M284K-hGALE further suggest that there are additional, stabilising factors present in the cell. Taken together, these results reinforce the need for care in the interpretation of in vitro, enzymatic diagnostic tests for type III galactosemia.     

＂Unconventional＂ Neutralizing Activity of Antibodies Against HIV

Neutralizing antibodies are recognized to be one of the essential elements of the adaptive immune response that must be induced by an effective vaccine against HIV. However,only a limited number of antibodies have been identified to neutralize a broad range of primary isolates of HIV-1 and attempts to induce such antibodies by immunization were unsuccessful. The difficulties to generate such antibodies are mainly due to intrinsic properties of HIV-1 envelope spikes,such as high sequence diversity,heavy glycosylation,and inducible and transient nature of certain epitopes. In vitro neutralizing antibodies are identified using "conventional" neutralization assay which uses phytohe-magglutinin (PHA)-stimulated human PBMCs as target cells. Thus,in essence the assay evaluates HIV-1 replication in CD4 T cells. Recently,several laboratories including us demonstrated that some monoclonal antibodies and HIV-1-specific polyclonal IgG purified from patient sera,although they do not have neutralizing activity when tested by the "conventional" neutralization assay,do exhibit potent and broad neutralizing activity in "unconventional" ways. The neutralizing activity of these antibodies and IgG fractions is acquired through post-translational modifications,through opsonization of virus particles into macrophages and immature dendritic cells (iDCs),or through expression of antibodies on the surface of HIV-1-susceptible cells. This review will focus on recent findings of this area and point out their potential applications in the development of preventive strategies against HIV.     

Calpain Inhibition Protects Spinal Motoneurons from the Excitotoxic Effects of AMPA In vivo

Microdialysis perfusion of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) in rat lumbar spinal cord produces severe motoneuron damage and consequently hindlimb paralysis. Here we studied the time course of the AMPA-induced neurodegenerative changes and motor alterations, and the protective effect of leupeptin, an inhibitor of calpain, a Ca(2+)-activated protease. Paralysis occurs at 4-6 h after AMPA perfusion, but cresyl violet staining showed that motoneuron damage starts at about 3 h and progresses until reaching 50% neuronal loss at 6 h and 90% loss at 12 h. In contrast, choline acetyltransferase (ChAT) immunohistochemistry revealed that the enzyme is already decreased at 30 min after AMPA perfusion and practically disappears at 3 h. Microdialysis coperfusion of leupeptin with AMPA prevented the motor alterations and paralysis and remarkably reduced both the decrement in ChAT immunoreactivity and the loss of motoneurons. We conclude that an increased Ca(2+) influx through Ca(2+)-permeable AMPA receptors activates calpain, and as a consequence ChAT content decreases earlier than other Ca(2+)-dependent processes, including the proteolytic activity of calpain, cause the death of motoneurons.     

Synthesis and In vitro Biological Activity of Cyclic Lipophilic χ-Conotoxin MrIA Analogues

The 13-residue peptide, χ-conotoxin MrIA extracted from the venom of Conus marmoreus, is a potent and selective inhibitor of the human noradrenaline transporter (NET). With the aim of improving its biophysical properties, chemical modifications were performed including the attachment of a lipophilic amino acid at the N-terminus and cyclisation of the peptide backbone with functionality introduced into the linker. All χ-conotoxin MrIA analogues were assembled on solid phase by highly optimised Boc chemistry and N- to C-cyclic analogues accessed by cysteine-mediated intramolecular native chemical ligation. In vitro biological activity at the human NET was evaluated by functional assays. All analogues inhibited the uptake of [³H]noradrenaline with comparable potencies to that of the native peptide, with one of the analogues, the linear N-terminal aminotetradecanoyl MrIA showing a 3-fold increase in potency (p < 0.05).     

Adverse Effect of Cyclosporin A on Barrier Functions of Cerebral Microvascular Endothelial Cells After Hypoxia-reoxygenation Damage In Vitro

Hypoxia and post-hypoxic reoxygenation induces disruption of the blood–brain barrier (BBB). Alterations of the BBB function after hypoxia/reoxygenation (H/R) injury remain unclear. Cyclosporin A (CsA), a potent immunosuppressant, induces neurotoxic effects by entering the brain, although the transport of CsA across the BBB is restricted by P-glycoprotein (P-gp), a multidrug efflux pump, and tight junctions of the brain capillary endothelial cells. The aim of this study was to evaluate whether the BBB after H/R damage is vulnerable to CsA-induced BBB dysfunction. We attempted to establish a pathophysiological BBB model with immortalized mouse brain capillary endothelial (MBEC4) cells. The effects of CsA on permeability and P-gp activity of the MBEC4 cells were then examined. Exposure to hypoxia for 4 h and reoxygenation for 1 h (H/R (4 h/1 h)) produced a significant decrease in P-gp function of MBEC4 cells, without changing cell viability and permeability for sodium fluorescein and Evan’s blue-albumin at 7 days after H/R (4 h/1 h). CsA-induced hyperpermeability and P-gp dysfunction in MBEC4 monolayers at 7 days after H/R (4 h/1 h) were exacerbated. The possibility that CsA penetrates the BBB with incomplete functions in the vicinity of cerebral infarcts to induce neurotoxicity has to be considered.     

In vivo species specificity of DNA polymerase α

The DNA polymerase a enzymes from human, and budding (Saccharomyces cerevisiae) and fission yeast (Schizosaccharomyces pombe) are homologous proteins involved in initiation and replication of chromosomal DNA. Sequence comparision of human DNA polymerase α with that of S. cerevisiae and S. pombe shows overall levels of amino acid sequence identity of 32% and 34%, respectively. We report here that, despite the sequence conservation among these three enzymes, functionally active human DNA polymerase a fails to rescue several different conditional lethal alleles of the budding yeast POL1 gene at nonpermissive temperature. Furthermore, human DNA polymerase α cannot complement a null allele of budding yeast POL1 either in germinating spores or in vegetatively growing cells. In fission yeast, functionally active human DNA polymerase α is also unable to complement the disrupted polα::ura4 ⁺ allele in germinating spores. Thus, in vivo, DNA polymerase α has stringent species specificity for initiation and replication of chromosomal DNA.     

In vivo response to the different LET regions of a 55 MeV helium ion beam

We evaluated the in vivo response to heavy particle irradiation in rat tail epidermis using silver-stained nucleolar organizer regions (AgNOR) as the end-point. The energy degradation of the beam across the circular section of the tail allowed us to study the damage elicited by two different LET regions of a helium beam, i.e. non-Bragg peak (NBP) and Bragg peak (BP), at different sites on the same sample. The tails were locally irradiated with a helium ion beam at different fluences. AgNOR exhibited marked variations between tissue areas only a few micrometers apart within each tail exposed to a given beam fluence. An analysis of the AgNOR variations in NBP and BP areas of tails exposed to different beam fluences revealed a dose-dependent effect. The AgNOR provide quantitative evidence of differential damage in neighboring tissue areas exposed to different LET regions of a helium-ion beam.     

In vivo uses of aptamers selected against cell surface biomarkers for therapy and molecular imaging

Nucleic acid Aptamers are ligands that are selected by a process of molecular evolution to bind with high affinities and specificities to a specific target. Recently, an increasing number of aptamers have been selected against biomarkers expressed at the surface of human cells or infectious pathogens. This class of targets, mostly proteins, is associated with several pathologies including cancer, inflammation and infection diseases. Several of these cell surface specific aptamers were tested in vivo as drugs or as targeting agents for nanocarriers, siRNA or contrast agents. Strikingly, they were used to develop a wide variety of new treatments or new approaches for molecular imaging and they were also able to improve current therapies such as chemotherapy, radiotherapy or immunotherapy. This review presents these different applications and the different studies conducted in vivo with this class of aptamers, predominantly in pre-clinical models.     

Microenvironmental influences of apoptosis in vivo and in vitro

The apoptosis program of physiological cell death elicits a range of non-phlogistic homeostatic mechanisms—“recognition, response and removal”—that regulate the microenvironments of normal and diseased tissues via multiple modalities operating over short and long distances. The molecular mechanisms mediate intercellular signaling through direct contact with neighboring cells, release of soluble factors and production of membrane-delimited fragments (apoptotic bodies, blebs and microparticles) that allow for interaction with host cells over long distances. These processes effect the selective recruitment of mononuclear phagocytes and the specific activation of both phagocytic and non-phagocytic cells. While much evidence is available concerning the mechanisms underlying the recognition and responses of phagocytes that culminate in the engulfment and removal of apoptotic cell bodies, relatively little is yet known about the non-phagocytic cellular responses to the apoptosis program. These responses regulate inflammatory and immune cell activation as well as cell fate decisions of proliferation, differentiation and death. Here, we review current knowledge of these processes, considering especially how apoptotic cells condition the microenvironments of normal and malignant tissues. We also discuss how apoptotic cells that persist in the absence of phagocytic clearance exert inhibitory effects over their viable neighbors, paying particular attention to the specific case of cell cultures and highlighting how new cell-corpse-clearance devices—Dead-Cert^® Nanoparticles—can significantly improve the efficacy of cell cultures through effective removal of non-viable cells in the absence of phagocytes in vitro.     

Phosphorylation of Recombinant Tristetraprolin In vitro

Tristetraprolin/zinc finger protein 36 (TTP/ZFP36) binds and destabilizes some proinflammatory cytokine mRNAs. TTP-deficient mice develop a profound inflammatory syndrome due to excessive production of proinflammatory cytokines. TTP gene expression is induced by various factors including insulin, cinnamon, and green tea extracts. Previous studies have shown that TTP is highly phosphorylated in vivo and multiple phosphorylation sites are identified in human TTP. This study evaluated the potential protein kinases that could phosphorylate recombinant TTP in vitro. Motif scanning suggested that TTP was a potential substrate for various kinases. SDS-PAGE showed that in vitro phosphorylation of TTP with p42 and p38 MAP kinases resulted in visible electrophoretic mobility shift of TTP to higher molecular masses. Autoradiography showed that TTP was phosphorylated in vitro by GSK3b, PKA, PKB, PKC, but not Cdc2, in addition to p42, p38, and JNK. These results demonstrate that TTP is a substrate for a number of protein kinases in vitro.     

Is Allelopathic Activity of Ipomoea murucoides Induced by Xylophage Damage?

Herbivory activates the synthesis of allelochemicals that can mediate plant-plant interactions. There is an inverse relationship between the activity of xylophages and the abundance of epiphytes on Ipomoea murucoides. Xylophagy may modify the branch chemical constitution, which also affects the liberation of allelochemicals with defense and allelopathic properties. We evaluated the bark chemical content and the effect of extracts from branches subjected to treatments of exclusion, mechanical damage and the presence/absence of epiphytes, on the seed germination of the epiphyte Tillandsia recurvata. Principal component analysis showed that branches without any treatment separate from branches subjected to treatments; damaged and excluded branches had similar chemical content but we found no evidence to relate intentional damage with allelopathy; however 1-hexadecanol, a defense volatile compound correlated positively with principal component (PC) 1. The chemical constitution of branches subject to exclusion plus damage or plus epiphytes was similar among them. PC2 indicated that palmitic acid (allelopathic compound) and squalene, a triterpene that attracts herbivore enemies, correlated positively with the inhibition of seed germination of T. recurvata. Inhibition of seed germination of T. recurvata was mainly correlated with the increment of palmitic acid and this compound reached higher concentrations in excluded branches treatments. Then, it is likely that the allelopathic response of I. murucoides would increase to the damage (shade, load) that may be caused by a high load of epiphytes than to damage caused by the xylophages.     

‘In vivo’ estimates of nitrate reductase activity

Summary A comparison was made of three different ways of preparing leaf material of Zea mays L. for the in vivo estimation of nitrate reductase activity. Slicing of lamineae into 1-mm strips gave the lowest overall activities and eliminated significant diurnal variation whilst the use of intact lamineae showed marked diurnal variation and the highest overall activity.