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Exogenous Addition (exogenous + addition)
Selected AbstractsHeparin-binding hemagglutinin (HBHA) of Mycobacterium leprae is expressed during infection and enhances bacterial adherence to epithelial cellsFEMS MICROBIOLOGY LETTERS, Issue 2 2009Cristiana Soares De Lima Abstract A heparin-binding hemagglutinin (HBHA) expressed on the surface of Mycobacterium tuberculosis is an antigenic protein that has been implicated in bacterial adherence to epithelial cells and systemic dissemination. In this study, the potential role of the Mycobacterium leprae HBHA (ML-HBHA) homologue in leprosy was investigated. Initially, the in vivo expression of HBHA and its association with the M. leprae cell envelope was confirmed by immunoblotting and proteomic analysis. Mycobacterium leprae recombinant HBHA (rML-HBHA) bound to a heparin,Sepharose column, and its capacity to act as an adhesin was demonstrated in experiments showing that the exogenous addition of the protein to latex beads or to M. leprae cells promotes a dramatic increase in association with epithelial cells. Finally, serum anti-HBHA immunoglobulin G levels were investigated in individuals infected with M. leprae. Altogether, our data indicate that HBHA is recognized during the course of bacterial infection in humans and may play a role in leprosy pathogenesis. [source] Radioiodinated clioquinol as a biomarker for ,-amyloid: Zn2+ complexes in Alzheimer's diseaseAGING CELL, Issue 1 2006Carlos Opazo Summary Neocortical ,-amyloid (A,) aggregates in Alzheimer's disease (AD) are enriched in transition metals that mediate assembly. Clioquinol (CQ) targets metal interaction with A, and inhibits amyloid pathology in transgenic mice. Here, we investigated the binding properties of radioiodinated CQ ([125I]CQ) to different in vitro and in vivo Alzheimer models. We observed saturable binding of [125I]CQ to synthetic A, precipitated by Zn2+ (Kd = 0.45 and 1.40 nm for A,1-42 and A,1-40, respectively), which was fully displaced by free Zn2+, Cu2+, the chelator DTPA (diethylene triamine pentaacetic acid) and partially by Congo red. Sucrose density gradient of post-mortem AD brain indicated that [125I]CQ concentrated in a fraction enriched for both A, and Zn, which was modulated by exogenous addition of Zn2+ or DTPA. APP transgenic (Tg2576) mice injected with [125I]CQ exhibited higher brain retention of tracer compared to non-Tg mice. Autoradiography of brain sections of these animals confirmed selective [125I]CQ enrichment in the neocortex. Histologically, both thioflavine-S (ThS)-positive and negative structures were labeled by [125I]CQ. A pilot SPECT study of [123I]CQ showed limited uptake of the tracer into the brain, which did however, appear to be more rapid in AD patients compared to age-matched controls. These data support metallated A, species as the neuropharmacological target of CQ and indicate that this drug class may have potential as in vivo imaging agents for Alzheimer neuropathology. [source] EFFECT OF VARIOUS ANTIOXIDANTS ON THE OXIDATIVE STABILITY OF ACID AND ALKALI SOLUBILIZED MUSCLE PROTEIN ISOLATESJOURNAL OF FOOD BIOCHEMISTRY, Issue 2 2009SIVAKUMAR RAGHAVAN ABSTRACT Protein isolates prepared from cod (Gadus morhua) myofibrillar proteins using acid or alkali solubilization are susceptible to oxidative rancidity. Oxidation could be delayed by the exogenous addition of antioxidants. The objective of this research was to compare the efficacy of antioxidants such as ,-tocopherol, butylated hydroxyanisole (BHA) and propyl gallate, to inhibit oxidation in acid- and alkali- solubilized cod protein isolates. Oxidation was catalyzed using cod hemolysate. Oxidation of lipids was monitored by the measurement of thiobarbituric acid reactive substances and painty odor. Results showed that protein isolates prepared using the acid process was significantly (P < 0.05) more susceptible to lipid oxidation than alkali-solubilized protein isolates. Regardless of pH treatments, the efficacy of various antioxidants decreased in the order propyl gallate > BHA > ,-tocopherol. PRACTICAL APPLICATIONS Research has shown that seafood available for human consumption is rapidly getting depleted and that many fish species may become extinct in the next half-century or so. Acid and alkali solubilization methods are recent but well-known techniques used for preparing protein isolates from under-utilized aquatic species and the by-products of seafood industry. Although numerous researchers have studied the use of acid and alkali processes on several sources of seafood, almost no research has been done on the use of antioxidants to protect protein isolates from lipid oxidation. In our research, we have studied the effect of various antioxidants on the oxidative stability of acid- and alkali-solubilized fish myofibrillar proteins. The results from this work will enable the seafood industry to properly identify the process and the type of antioxidants required for making muscle food products with increased oxidative stability. [source] Study of the Biosynthesis of 1-Octen-3-ol Using a Crude Homogenate of Agaricus bisporus in a BioreactorJOURNAL OF FOOD SCIENCE, Issue 3 2008R.O. Morawicki ABSTRACT:, 1-Octen-3-ol and 10-oxo- trans -8-decenoic acid are metabolites of the breakdown of linoleic acid (LA) by mushroom enzymes. These compounds can be produced in a bioreactor using a crude mushroom homogenate and the exogenous addition of LA and oxygen. The factors' duration of blending, mushroom,buffer ratio, effect of a surfactant, whole against partially clarified reaction broths, purity of LA, and utilization of stumps instead of whole mushrooms were studied for their effect on reaction yield using a 1-L bioreactor. The results showed the feasibility of using the more inexpensive 60%-pure LA instead of the 99%-pure LA even when a yield loss was involved. Waste stumps could be used instead of whole mushrooms with a yield decline of 26%. [source] Functional expression of corticotropin-releasing hormone (CRH) receptor 1 in cultured rat microgliaJOURNAL OF NEUROCHEMISTRY, Issue 2 2002Wei Wang Abstract Corticotropin-releasing hormone (CRH), known as a key regulator of the hypothalamic,pituitary,adrenal axis response to stress, elicits its biological effects by binding to two membrane receptors (CRH-R1 and CRH-R2). The present studies examined the presence of functional expression of CRH receptors in cultured microglia of rat. CRH-R1 mRNA and protein were detected by reverse transcriptase polymerase chain reaction (RT-PCR), western blotting and receptor chemical cross-linking assay in cultured microglia. CRH-R2 mRNA was undectable by RT-PCR. The radioligand binding analysis using [125I]Tyr-rat/human CRH revealed a high affinity binding site (Kd of 1.2 nm and Bmax of 84 fmol/mg of protein). Competition studies using CRH and related peptides indicated kinetic and pharmacological characteristics consistent with the CRH-R1 receptor subtype. Receptor chemical cross-linking assay demonstrated a single band of CRH receptor with a molecular weight of ,77 kDa, which was inhibited in the presence of excess unlabeled rat/human CRH in a dose-dependent manner and inhibited by a CRH receptor,antagonist astressin. Functional coupled cAMP production in cultured microglia was stimulated by exogenous addition of CRH and related peptides in a dose-dependent manner and blocked by astressin. Our findings suggest the functional expression of CRH-R1 receptor in rat microglia, indicating an important mechanism of interaction between immune and neuroendocrine systems in brain physiological and,pathological conditions. [source] Dysregulation of human bestrophin-1 by ceramide-induced dephosphorylationTHE JOURNAL OF PHYSIOLOGY, Issue 18 2009Qinghuan Xiao Best vitelliform macular dystrophy is an inherited autosomal dominant, juvenile onset form of macular degeneration caused by mutations in a chloride ion channel, human bestrophin-1 (hBest1). Mutations in Best1 have also been linked to several other forms of retinopathy. In addition to mutations, hBest1 dysfunction might come about by disruption of other processes that regulate Best1 function. Here we show that hBest1 chloride channel activity is regulated by ceramide and phosphorylation. We have identified a protein kinase C (PKC) phosphorylation site (serine 358) in hBest1 that is important for sustained channel function. Channel activity is maintained by PKC activators, protein phosphatase inhibitors, or pseudo-phosphorylation by substitution of glutamic acid for serine 358. When ceramide levels are elevated by exogenous addition of ceramide to the bath, by addition of bacterial sphingomyelinase, or by hypertonic stress, S358 is rapidly dephosphorylated. The dephosphorylation is mediated by protein phosphatase 2A. Hypertonic stress-induced dephosphorylation is blocked by a dihydroceramide, an inactive form of ceramide, and manumycin, an inhibitor of neutral sphingomyelinase. Our results support a model in which ceramide accumulation during early stages of retinopathy inhibits hBest1 function, leading to abnormal fluid transport across the retina, and enhanced inflammation. [source] Cytokinins negatively regulate the root iron uptake machinery in Arabidopsis through a growth-dependent pathwayTHE PLANT JOURNAL, Issue 2 2008Mathilde Séguéla Summary Plants display a number of biochemical and developmental responses to low iron availability in order to increase iron uptake from the soil. The ferric-chelate reductase FRO2 and the ferrous iron transporter IRT1 control iron entry from the soil into the root epidermis. In Arabidopsis, expression of IRT1 and FRO2 is tightly controlled to maintain iron homeostasis, and involves local and long-distance signals, as well as transcriptional and post-transcriptional events. FIT encodes a putative basic helix-loop-helix (bHLH) transcription factor that regulates iron uptake responses in Arabidopsis. Here, we uncover a new regulation of the root iron uptake genes. We show that IRT1, FRO2 and FIT are repressed by the exogenous addition of cytokinins (CKs), and that this repression acts at the level of transcript accumulation, and depends on the AHK3 and CRE1 CK receptors. The CKs and iron-deficiency signals act through distinct pathways to regulate the soil iron uptake genes, as (i) CK repression is independent of the iron status, (ii) IRT1 and FRO2 downregulation is unchanged in a fit loss-of-function mutant, indicating that FIT does not mediate CK repression, and (iii) the iron-regulated genes AtNRAMP3 and AtNRAMP4 are not downregulated by CKs. We show that root growth-inhibitory conditions, such as abiotic stresses (mannitol, NaCl) and hormonal treatments (auxin, abscissic acid), repress the iron starvation response genes. We propose that CKs control the root iron uptake machinery through a root growth dependent pathway in order to adapt nutrient uptake to the demand of the plant. [source] Chloroplast signalling in the light induction of nuclear HSP70 genes requires the accumulation of chlorophyll precursors and their accessibility to cytoplasm/nucleusTHE PLANT JOURNAL, Issue 4 2000Janette Kropat Summary Chlorophyll precursors Mg-protoporphyrin IX and its monomethylester are candidates for plastid-derived molecules involved in light signalling from the chloroplast to the nucleus. The pool sizes of these two Mg2+ -containing porphyrins and of protoporphyrin IX transiently increased upon a shift of Chlamydomonas cultures from dark to light. This increase coincided with the accumulation of mRNAs encoded by the nuclear genes HSP70A and HSP70B. Analysis of a mutant (brs-1), previously shown to be defective in the light induction of these genes, revealed high levels of protoporphyrin IX but no light-induced increase in the levels of Mg2+ -containing porphyrins. Inhibitors of cytoplasmic protein synthesis prevented both the light-induced rise in pool levels and induction of the HSP70 genes. Similarly, pre-gametes, intermediates of sexual differentiation, lacked both responses to light. The block in light induction of the HSP70 genes in inhibitor-treated cells and in pre-gametes could be circumvented by the exogenous addition of Mg-protoporphyrin IX in the dark. This suggests an essential role for light-induced Mg-protoporphyrin IX accumulation in this chloroplast-to-nucleus signalling pathway. However, accumulation of this porphyrin in the dark , presumably in the chloroplast , did not result in induction. A second crucial role for light in this signalling pathway is postulated which makes this plastidic compound accessible to the cytoplasm/nucleus where the downstream signalling pathway may be activated. [source] Switchgrass leaching requirements for solid-state fermentation by Acidothermus cellulolyticusBIOTECHNOLOGY PROGRESS, Issue 3 2010Jean S. VanderGheynst Abstract Growth of Acidothermus cellulolyticus in solid-state fermentation and its required growth conditions were investigated in this study. Extraction of switchgrass was required for growth. Under the experimental conditions, extraction ratio had the most significant effect on the growth of A. cellulolyticus. Heat treatment (in the form of autoclaving) of switchgrass did not have a significant effect on the growth rate; however, longer heat treatment times had a negative effect on the total growth. Moisture content adjustment had no effect on the release of inhibitors into extracts. Our results showed that leaching at a minimum 40:1 (gram water: gram dry biomass) removed inhibitory compound(s) from switchgrass. Upon extraction A. cellulolyticus colonized switchgrass in solid fermentation without exogenous addition of carbon and nitrogen sources. It is the first demonstration of growth of A. cellulolyticus in solid fermentation. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010 [source] Enzymatic Redox Cofactor Regeneration in Organic Media: Functionalization and Application of Glycerol Dehydrogenase and Soluble Transhydrogenase in Reverse MicellesBIOTECHNOLOGY PROGRESS, Issue 4 2005Hirofumi Ichinose An enzymatic system for the regeneration of redox cofactors NADH and NADPH was investigated in nanostructural reverse micelles using bacterial glycerol dehydrogenase (GLD) and soluble transhydrogenase (STH). Catalytic conversion of NAD+ to NADH was realized in the sodium dioctylsulfosuccinate (AOT)/isooctane reverse micellar system harboring GLD and a sacrificial substrate, glycerol. The initial rate of NADH regeneration was enhanced by exogenous addition of ammonium sulfate into the reverse micelles, suggesting that NH4+ acts as a monovalent cationic activator. STH was successfully entrapped in the AOT/isooctane reverse micelles as well as GLD and was revealed to be capable of catalyzing the stoichiometric hydrogen transfer reaction between NADP+ and NADPH in reverse micelles. These results indicate that GLD and STH have potential for use in redox cofactor recycling in reverse micelles, which allows the use of catalytic quantities of NAD(P)H in organic media. [source] Effects of exogenous fatty acids and cholesterol on aminopeptidase activities in rat astrogliaCELL BIOCHEMISTRY AND FUNCTION, Issue 4 2002M. J. Ramírez-Expósito Abstract Several studies have addressed the interaction between fatty acids and lipids with central nervous system peptides. Because aminopeptidases (AP) are involved in the regulation of neuropeptides, this work studies several AP expressed in cultured astroglia, after exogenous addition of oleic and linoleic fatty acids and cholesterol to the culture medium. Alanyl-AP, arginyl-AP, cystyl-AP, leucyl-AP, tyrosyl-AP and pyroglutamyl-AP activities were analysed in whole cells using the corresponding aminoacyl-,-naphthylamides as substrates. Oleic acid inhibits alanyl-AP, cystyl-AP and leucyl-AP activities, whereas linoleic acid inhibits alanyl-AP, arginyl-AP and tyrosyl-AP activities. Neither oleic acid nor linoleic acid modifies pyroglutamyl-AP activity. In contrast, cholesterol increases arginyl-AP, cystyl-AP, leucyl-AP, tyrosyl-AP and pyroglutamyl-AP activities, although it does not modify alanyl-AP activity. The changes reported here suggest that oleic and linoleic fatty acids and cholesterol can modulate peptide activities via their degradation route involving aminopeptidases; each of them being differentially regulated. Copyright © 2002 John Wiley & Sons, Ltd. [source] | |||||||||||||