Two pheromone precursor genes are transcriptionally expressed in the homothallic ascomycete Sordaria macrospora.

PubMed

Pöggeler, S

2000-06-01

In order to analyze the involvement of pheromones in cell recognition and mating in a homothallic fungus, two putative pheromone precursor genes, named ppg1 and ppg2, were isolated from a genomic library of Sordaria macrospora. The ppg1 gene is predicted to encode a precursor pheromone that is processed by a Kex2-like protease to yield a pheromone that is structurally similar to the alpha-factor of the yeast Saccharomyces cerevisiae. The ppg2 gene encodes a 24-amino-acid polypeptide that contains a putative farnesylated and carboxy methylated C-terminal cysteine residue. The sequences of the predicted pheromones display strong structural similarity to those encoded by putative pheromones of heterothallic filamentous ascomycetes. Both genes are expressed during the life cycle of S. macrospora. This is the first description of pheromone precursor genes encoded by a homothallic fungus. Southern-hybridization experiments indicated that ppg1 and ppg2 homologues are also present in other homothallic ascomycetes.

Activin A stimulates IkappaB-alpha/NFkappaB and RANK expression for osteoclast differentiation, but not AKT survival pathway in osteoclast precursors.

PubMed

Sugatani, T; Alvarez, U M; Hruska, K A

2003-09-01

Recent studies have reported that activin A enhances osteoclastogenesis in cultures of mouse bone marrow cells stimulated with receptor activator of nuclear factor-kappaB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). However, the exact mechanisms by which activin A functions during osteoclastogenesis are not clear. RANKL stimulation of RANK/TRAF6 signaling increases nuclear factor-kappaB (NFkappaB) nuclear translocation and activates the Akt/PKB cell survival pathway. Here we report that activin A alone activates IkappaB-alpha, and stimulates nuclear translocation of NFkappaB and receptor activator of nuclear factor-kappaB (RANK) expression for osteoclastogenesis, but not Akt/PKB survival signal transduction including BAD and mammalian target of rapamycin (mTOR) for survival in osteoclast precursors in vitro. Activin A alone failed to activate Akt, BAD, and mTOR by immunoblotting, and it also failed to prevent apoptosis in osteoclast precursors. While activin A activated IkappaB-alpha and induced nuclear translocation of phosphorylated-NFkappaB, and it also enhanced RANK expression in osteoclast precursors. Moreover, activin A enhanced RANKL- and M-CSF-stimulated nuclear translocation of NFkappaB. Our data suggest that activin A enhances osteoclastogenesis treated with RANKL and M-CSF via stimulation of RANK, thereby increasing the RANKL stimulation. Activin A alone activated the NFkappaB pathway, but not survival in osteoclast precursors in vitro, but it is, thus, insufficient as a sole stimulus to osteoclastogenesis. Copyright 2003 Wiley-Liss, Inc.

Structure and regulation of KGD1, the structural gene for yeast alpha-ketoglutarate dehydrogenase.

PubMed

Repetto, B; Tzagoloff, A

1989-06-01

Nuclear respiratory-defective mutants of Saccharomyces cerevisiae have been screened for lesions in the mitochondrial alpha-ketoglutarate dehydrogenase complex. Strains assigned to complementation group G70 were ascertained to be deficient in enzyme activity due to mutations in the KGD1 gene coding for the alpha-ketoglutarate dehydrogenase component of the complex. The KGD1 gene has been cloned by transformation of a representative kgd1 mutant, C225/U1, with a recombinant plasmid library of wild-type yeast nuclear DNA. Transformants containing the gene on a multicopy plasmid had three- to four-times-higher alpha-ketoglutarate dehydrogenase activity than did wild-type S. cerevisiae. Substitution of the chromosomal copy of KGD1 with a disrupted allele (kgd1::URA3) induced a deficiency in alpha-ketoglutarate dehydrogenase. The sequence of the cloned region of DNA which complements kgd1 mutants was found to have an open reading frame of 3,042 nucleotides capable of coding for a protein of Mw 114,470. The encoded protein had 38% identical residues with the reported sequence of alpha-ketoglutarate dehydrogenase from Escherichia coli. Two lines of evidence indicated that transcription of KGD1 is catabolite repressed. Higher steady-state levels of KGD1 mRNA were detected in wild-type yeast grown on the nonrepressible sugar galactose than in yeast grown on high glucose. Regulation of KGD1 was also studied by fusing different 5'-flanking regions of KGD1 to the lacZ gene of E. coli and measuring the expression of beta-galactosidase in yeast. Transformants harboring a fusion of 693 nucleotides of the 5'-flanking sequence expressed 10 times more beta-galactosidase activity when grown under derepressed conditions. The response to the carbon source was reduced dramatically when the same lacZ fusion was present in a hap2 or hap3 mutant. The promoter element(s) responsible for the regulated expression of KGD1 has been mapped to the -354 to -143 region. This region contained several

Tumour necrosis factor-alpha impairs neuronal differentiation but not proliferation of hippocampal neural precursor cells: Role of Hes1.

PubMed

Keohane, Aoife; Ryan, Sinead; Maloney, Eimer; Sullivan, Aideen M; Nolan, Yvonne M

2010-01-01

Tumour necrosis factor-alpha (TNFalpha) is a pro-inflammatory cytokine, which influences neuronal survival and function yet there is limited information available on its effects on hippocampal neural precursor cells (NPCs). We show that TNFalpha treatment during proliferation had no effect on the percentage of proliferating cells prepared from embryonic rat hippocampal neurosphere cultures, nor did it affect cell fate towards either an astrocytic or neuronal lineage when cells were then allowed to differentiate. However, when cells were differentiated in the presence of TNFalpha, significantly reduced percentages of newly born and post-mitotic neurons, significantly increased percentages of astrocytes and increased expression of TNFalpha receptors, TNF-R1 and TNF-R2, as well as expression of the anti-neurogenic Hes1 gene, were observed. These data indicate that exposure of hippocampal NPCs to TNFalpha when they are undergoing differentiation but not proliferation has a detrimental effect on their neuronal lineage fate, which may be mediated through increased expression of Hes1. Copyright 2009 Elsevier Inc. All rights reserved.

Viral Repression of Fungal Pheromone Precursor Gene Expression

PubMed Central

Zhang, Lei; Baasiri, Rudeina A.; Van Alfen, Neal K.

1998-01-01

Biological control of chestnut blight caused by the filamentous ascomycete Cryphonectria parasitica can be achieved with a virus that infects this fungus. This hypovirus causes a perturbation of fungal development that results in low virulence (hypovirulence), poor asexual sporulation, and female infertility without affecting fungal growth in culture. At the molecular level, the virus is known to affect the transcription of a number of fungal genes. Two of these genes, Vir1 and Vir2, produce abundant transcripts in noninfected strains of the fungus, but the transcripts are not detectable in virus-infected strains. We report here that these two genes encode the pheromone precursors of the Mat-2 mating type of the fungus; consequently, these genes have been renamed Mf2/1 and Mf2/2. To determine if the virus affects the mating systems of both mating types of this fungus, the pheromone precursor gene, Mf1/1, of a Mat-1 strain was cloned and likewise was found to be repressed in virus-infected strains. The suppression of transcription of the pheromone precursor genes of this fungus could be the cause of the mating defect of infected strains of the fungus. Although published reports suggest that a Gαi subunit may be involved in this regulation, our results do not support this hypothesis. The prepropheromone encoded by Mf1/1 is structurally similar to that of the prepro-p-factor of Schizosaccharomyces pombe. This is the first description of the complete set of pheromone precursor genes encoded by a filamentous ascomycete. PMID:9447992

The central domain of yeast transcription factor Rpn4 facilitates degradation of reporter protein in human cells.

PubMed

Morozov, A V; Spasskaya, D S; Karpov, D S; Karpov, V L

2014-10-16

Despite high interest in the cellular degradation machinery and protein degradation signals (degrons), few degrons with universal activity along species have been identified. It has been shown that fusion of a target protein with a degradation signal from mammalian ornithine decarboxylase (ODC) induces fast proteasomal degradation of the chimera in both mammalian and yeast cells. However, no degrons from yeast-encoded proteins capable to function in mammalian cells were identified so far. Here, we demonstrate that the yeast transcription factor Rpn4 undergoes fast proteasomal degradation and its central domain can destabilize green fluorescent protein and Alpha-fetoprotein in human HEK 293T cells. Furthermore, we confirm the activity of this degron in yeast. Thus, the Rpn4 central domain is an effective interspecies degradation signal. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

[Engineering of a System for the Production of Mutant Human Alpha-Fetoprotein in the Methylotrophic Yeast Pichia pastoris].

PubMed

Morozkina, E V; Vavilova, E A; Zatsepin, S S; Klyachko, E V; Yagudin, T A; Chulkin, A M; Dudich, I V; Semenkova, L N; Churilova, I V; Benevolenskii, S V

2016-01-01

A system for the production of mutant recombinant human alpha-fetoprotein (rhAFPO) lacking the glycosylation site has been engineered in the yeast Pichia pastoris. A strain of the methylotrophic yeast Pichia pastoris GS 115/pPICZ?A/rhAFP0, which produces unglycosylated rhAFPO and secretes it to the culture medium, has been constructed. Optimization and scale-up of the fermentation technology have resulted in an increase in the rhAFP0 yield to 20 mg/L. A scheme of isolation and purification of biologically active rhAFP0 has been developed. The synthesized protein has the antitumor activity, which is analogous to the activity of natural human embryonic alpha-fetoprotein.

The yeast genome may harbor hypoxia response elements (HRE).

PubMed

Ferreira, Túlio César; Hertzberg, Libi; Gassmann, Max; Campos, Elida Geralda

2007-01-01

The hypoxia-inducible factor-1 (HIF-1) is a heterodimeric transcription factor activated when cells are submitted to hypoxia. The heterodimer is composed of two subunits, HIF-1alpha and the constitutively expressed HIF-1beta. During normoxia, HIF-1alpha is degraded by the 26S proteasome, but hypoxia causes HIF-1alpha to be stabilized, enter the nucleus and bind to HIF-1beta, thus forming the active complex. The complex then binds to the regulatory sequences of various genes involved in physiological and pathological processes. The specific regulatory sequence recognized by HIF-1 is the hypoxia response element (HRE) that has the consensus sequence 5'BRCGTGVBBB3'. Although the basic transcriptional regulation machinery is conserved between yeast and mammals, Saccharomyces cerevisiae does not express HIF-1 subunits. However, we hypothesized that baker's yeast has a protein analogous to HIF-1 which participates in the response to changes in oxygen levels by binding to HRE sequences. In this study we screened the yeast genome for HREs using probabilistic motif search tools. We described 24 yeast genes containing motifs with high probability of being HREs (p-value<0.1) and classified them according to biological function. Our results show that S. cerevisiae may harbor HREs and indicate that a transcription factor analogous to HIF-1 may exist in this organism.

Utilization of alpha-ketoglutarate as a precursor for transmitter glutamate in cultured cerebellar granule cells.

PubMed

Peng, L A; Schousboe, A; Hertz, L

1991-01-01

Alpha-ketoglutarate together with an amino group donor (alanine) was shown to be able to serve as a precursor for the glutamate pool which is released by potassium-induced depolarization (i.e., transmitter glutamate) in cerebellar granule cells. However, these compounds could not be utilized as precursors for intracellular glutamate or for release of transmitter aspartate. The formation of transmitter glutamate was inhibited by the transamination inhibitor aminooxyacetic acid but not by phenylsuccinate, an inhibitor of the dicarboxylate carrier in the mitochondrial membrane. Both of these inhibitors have previously been found to inhibit synthesis of transmitter glutamate from glutamine. The results support the hypothesis that alpha-ketoglutarate and alanine undergo transmination in the cytosol to form pyruvate and glutamate, and that this glutamate pool is available for transmitter release of glutamate but does not constitute the major intracellular pool of glutamate.

Meat flavor precursors and factors influencing flavor precursors--A systematic review.

PubMed

Khan, Muhammad Issa; Jo, Cheorun; Tariq, Muhammad Rizwan

2015-12-01

Flavor is the sensory impression sensed by taste and smell buds and is a leading factor determining the meat quality and purchasing decision of the consumer. Meat flavor is characteristic of volatiles produced as a result of reactions of non-volatile components that are induced thermally. The water soluble compounds having low molecular weight and meat lipids are important precursors of cooked meat flavor. The Maillard reaction, lipid oxidation, and vitamin degradation are leading reactions during cooking which develop meat flavor from uncooked meat with little aroma and bloody taste. The pre-slaughter and postmortem factors like animal breed, sex, age, feed, aging and cooking conditions contribute to flavor development of cooked meat. The objective of this review is to highlight the flavor chemistry, meat flavor precursors and factors affecting meat flavor precursors. Copyright © 2015 Elsevier Ltd. All rights reserved.

Processing of intervening sequences: a new yeast mutant which fails to excise intervening sequences from precursor tRNAs.

PubMed

Hopper, A K; Schultz, L D; Shapiro, R A

1980-03-01

By using conditional loss of suppression an an assay, we have been successful in screening for a yeast mutant which is defective in tRNA processing. The los1-1 mutation causes an accumulation of a subset of precursor tRNAs at the nonpermissive temperature. These pre-tRNAs are like those which accumulate in the yeast mutant ts 136 (rna1) in that they have transcribed intervening sequences. The mutations at los1-1 and rna1 complement and segregate independently of each other. The los1-1 mutation affects the expression of all 8 tyrosine-inserting suppressor loci, but does not seem to affect rRNA or mRNA synthesis.

Increased neuronal beta-amyloid precursor protein expression in human temporal lobe epilepsy: association with interleukin-1 alpha immunoreactivity.

PubMed

Sheng, J G; Boop, F A; Mrak, R E; Griffin, W S

1994-11-01

Levels of immunoreactive beta-amyloid precursor protein and interleukin-1 alpha were found to be elevated in surgically resected human temporal lobe tissue from patients with intractable epilepsy compared with postmortem tissue from neurologically unaffected patients (controls). In tissue from epileptics, the levels of the 135-kDa beta-amyloid precursor protein isoform were elevated to fourfold (p < 0.05) those of controls and those of the 130-kDa isoform to threefold (p < 0.05), whereas those of the 120-kDa isoform (p > 0.05) were not different from control values. beta-Amyloid precursor protein-immunoreactive neurons were 16 times more numerous, and their cytoplasm and proximal processes were more intensely immunoreactive in tissue sections from epileptics than controls (133 +/- 12 vs. 8 +/- 3/mm2; p < 0.001). However, neither beta-amyloid precursor protein-immunoreactive dystrophic neurites nor beta-amyloid deposits were found in this tissue. Interleukin-1 alpha-immunoreactive cells (microglia) were three times more numerous in epileptics than in controls (80 +/- 8 vs. 25 +/- 5/mm2; p < 0.001), and these cells were often found adjacent to beta-amyloid precursor protein-immunoreactive neuronal cell bodies. Our findings, together with functions established in vitro for interleukin-1, suggest that increased expression of this protein contributes to the increased levels of beta-amyloid precursor protein in epileptics, thus indicating a potential role for both of these proteins in the neuronal dysfunctions, e.g., hyperexcitability, characteristic of epilepsy.

Human alpha beta hydrolase domain containing protein 11 and its yeast homolog are lipid hydrolases

DOE Office of Scientific and Technical Information (OSTI.GOV)

Arya, Madhuri; Srinivasan, Malathi; Rajasekharan, Ram

Mammalian alpha/beta hydrolase domain (ABHD) family of proteins have emerged as key regulators of lipid metabolism and are found to be associated with human diseases. Human α/β-hydrolase domain containing protein 11 (ABHD11) has recently been predicted as a potential biomarker for human lung adenocarcinoma. In silico analyses of the ABHD11 protein sequence revealed the presence of a conserved lipase motif GXSXG. However, the role of ABHD11 in lipid metabolism is not known. To understand the biological function of ABHD11, we heterologously expressed the human ABHD11 in budding yeast, Saccharomyces cerevisiae. In vivo [{sup 14}C]acetate labeling of cellular lipids in yeast cellsmore » overexpressing ABHD11 showed a decrease in triacylglycerol content. Overexpression of ABHD11 also alters the molecular species of triacylglycerol in yeast. Similar activity was observed in its yeast homolog, Ygr031w. The role of the conserved lipase motif in the hydrolase activity was proven by the mutation of all conserved amino acid residues of GXSXG motif. Collectively, our results demonstrate that human ABHD11 and its yeast homolog YGR031W have a pivotal role in the lipid metabolism. - Highlights: • Overexpression of ABHD11 protein and its yeast homolog Ygr031w cause a reduction in triacylglycerol levels in yeast. • The reduction in triacylglycerol is due to the presence of lipase motif GXSXG. • Overexpression of ABHD11 and Ygr031w alters the molecular species of triacylglycerol.« less

Structure, synthesis, and molecular cloning of dermaseptins B, a family of skin peptide antibiotics.

PubMed

Charpentier, S; Amiche, M; Mester, J; Vouille, V; Le Caer, J P; Nicolas, P; Delfour, A

1998-06-12

Analysis of antimicrobial activities that are present in the skin secretions of the South American frog Phyllomedusa bicolor revealed six polycationic (lysine-rich) and amphipathic alpha-helical peptides, 24-33 residues long, termed dermaseptins B1 to B6, respectively. Prepro-dermaseptins B all contain an almost identical signal peptide, which is followed by a conserved acidic propiece, a processing signal Lys-Arg, and a dermaseptin progenitor sequence. The 22-residue signal peptide plus the first 3 residues of the acidic propiece are encoded by conserved nucleotides encompassed by the first coding exon of the dermaseptin genes. The 25-residue amino-terminal region of prepro-dermaseptins B shares 50% identity with the corresponding region of precursors for D-amino acid containing opioid peptides or for antimicrobial peptides originating from the skin of distantly related frog species. The remarkable similarity found between prepro-proteins that encode end products with strikingly different sequences, conformations, biological activities and modes of action suggests that the corresponding genes have evolved through dissemination of a conserved "secretory cassette" exon.

The cytoplasmic end of transmembrane domain 3 regulates the activity of the Saccharomyces cerevisiae G-protein-coupled alpha-factor receptor.

PubMed Central

Parrish, William; Eilers, Markus; Ying, Weiwen; Konopka, James B

2002-01-01

The binding of alpha-factor to its receptor (Ste2p) activates a G-protein-signaling pathway leading to conjugation of MATa cells of the budding yeast S. cerevisiae. We conducted a genetic screen to identify constitutively activating mutations in the N-terminal region of the alpha-factor receptor that includes transmembrane domains 1-5. This approach identified 12 unique constitutively activating mutations, the strongest of which affected polar residues at the cytoplasmic ends of transmembrane domains 2 and 3 (Asn84 and Gln149, respectively) that are conserved in the alpha-factor receptors of divergent yeast species. Targeted mutagenesis, in combination with molecular modeling studies, suggested that Gln149 is oriented toward the core of the transmembrane helix bundle where it may be involved in mediating an interaction with Asn84. These residues appear to play specific roles in maintaining the inactive conformation of the protein since a variety of mutations at either position cause constitutive receptor signaling. Interestingly, the activity of many mammalian G-protein-coupled receptors is also regulated by conserved polar residues (the E/DRY motif) at the cytoplasmic end of transmembrane domain 3. Altogether, the results of this study suggest a conserved role for the cytoplasmic end of transmembrane domain 3 in regulating the activity of divergent G-protein-coupled receptors. PMID:11861550

In vitro studies on the translocation of acid phosphatase into the endoplasmic reticulum of the yeast Saccharomyces cerevisiae.

PubMed

Krebs, H O; Hoffschulte, H K; Müller, M

1989-05-01

We demonstrate here the in vitro translocation of yeast acid phosphatase into rough endoplasmic reticulum. The precursor of the repressible acid phosphatase from Saccharomyces cerevisiae encoded by the PHO5 gene, was synthesized in a yeast lysate programmed with in vitro transcribed PHO5 mRNA. In the presence of yeast rough microsomes up to 16% of the acid phosphatase synthesized was found to be translocated into the microsomes, as judged by proteinase resistance, and fully core-glycosylated. The translocation efficiency however, decreased to 3% if yeast rough microsomes were added after synthesis of acid phosphatase had been terminated. When a wheat-germ extract was used for in vitro synthesis, the precursor of acid phosphatase was translocated into canine pancreatic rough microsomes and thereby core-glycosylated in a signal-recognition-particle-dependent manner. Replacing canine with yeast rough microsomes in the wheat-germ translation system, however, resulted in a significant decrease in the ability to translocate and glycosylate the precursor. Translocation and glycosylation were partially restored by a high-salt extract prepared from yeast ribosomes. The results presented here suggest that yeast-specific factors are needed to translocate and glycosylate acid phosphatase efficiently in vitro.

Further studies on the quaternary structure of yeast casein kinase II.

PubMed

Szyszka, R; Lopaczyński, W; Gałasiński, W; Grankowski, N; Gasior, E

1986-01-01

Casein kinase type II were isolated by the same procedure, from rat liver, human placenta, Querin carcinoma and yeast, and characterized. The mammalian enzymes were composed of three subunits alpha, alpha' and beta, whereas yeast kinase was composed of two subunits alpha and alpha'. It was shown that the catalytic activity, substrate and phosphate donor specificity, sensitivity to heparin and spermine were the same for all the kinases tested. The results give additional support to the suggestion [1] that the beta subunit is not required for optimal activity and specificity of yeast casein kinase II. The quaternary structure of the yeast enzyme of a molecular weight of approximately 150 000 is proposed as alpha2 alpha'2.

Expression of the transforming growth factor alpha protooncogene in differentiating human promyelocytic leukemia (HL-60) cells.

PubMed

Walz, T M; Malm, C; Wasteson, A

1993-01-01

The process of myeloid differentiation in human promyelocytic leukemia cells (HL-60) is accompanied by the coordinate expression of numerous protooncogenes. To investigate the expression of transforming growth factor alpha (TGF-alpha) in myeloid differentiation, HL-60 cells were induced to differentiate into granulocytes with 1.25% dimethyl sulfoxide, 0.2 microM all-trans retinoic acid, or 500 microM N6,O2-dibutyryladenosine-3'5'-cyclic monophosphate or differentiated along the monocyte/macrophage pathway with 0.1 microM phorbol-12-myristate-13-acetate. Using Northern blot analyses, TGF-alpha transcripts were detected within 24 h of treatment in cells differentiating toward granulocytes; maximal levels of gene expression were reached after 3 days or later and remained essentially constant throughout the observation period. These cells released TGF-alpha protein, as demonstrated by analysis of the incubation medium. In contrast, no TGF-alpha RNA or protein was detectable in HL-60 cell cultures when induced with phorbol-12-myristate-13-acetate. Epidermal growth factor receptor transcripts could not be detected either in undifferentiated or in differentiated HL-60 cells; therefore it appears as if an autocrine loop involving TGF-alpha in HL-60 cells is unlikely. In conclusion, the results demonstrate, for the first time, the expression of TGF-alpha in human granulocyte precursor cells. Our findings may indicate novel regulatory pathways in hematopoiesis.

In vitro and in vivo expression of interleukin-1alpha and tumor necrosis factor-alpha mRNA in pemphigus vulgaris: interleukin-1alpha and tumor necrosis factor-alpha are involved in acantholysis.

PubMed

Feliciani, C; Toto, P; Amerio, P; Pour, S M; Coscione, G; Shivji, G; Wang, B; Sauder, D N

2000-01-01

Keratinocyte-derived cytokines have been implicated in the pathogenesis of a number of skin diseases. In this study we examined the possible role of keratinocyte-derived cytokines in the development of acantholysis in pemphigus vulgaris. Nineteen patients with pemphigus vulgaris, demonstrating the characteristic clinical, pathologic, and immunopathologic findings were studied. In situ immunolabeling demonstrated the presence of two cytokines interleukin-1alpha and tumor necrosis factor-alpha, in lesional and perilesional areas. Results were confirmed by reverse transcriptase-polymerase chain reaction, demonstrating overexpression of both cytokines in vivo. To study the role of these cytokines in the pathogenesis of pemphigus vulgaris both in vitro and in vivo studies were performed. The results of the in vitro study demonstrated that pemphigus vulgaris IgG induced interleukin-1alpha and tumor necrosis factor-alpha mRNA in the skin. The potential pathogenic role of these mediators was demonstrated by a blocking study using antibodies against human interleukin-1alpha and tumor necrosis factor-alpha in keratinocytes cultures. A combination of anti-interleukin-1alpha and anti-tumor necrosis factor-alpha antibodies inhibited in vitro pemphigus vulgaris IgG induced acantholysis. To confirm the role of interleukin-1 and tumor necrosis factor-alpha in pemphigus, we utilized passive transfer studies using interleukin-1 deficient mice (ICE-/-, interleukin-1beta-/-) and tumor necrosis factor-alpha receptor deficient mice (TNFR1R2-/-). Both groups demonstrated a decreased susceptibility to the passive transfer of pemphigus. Our data support the role of cytokines interleukin-1 and tumor necrosis factor-alpha in the pathogenesis of pemphigus vulgaris.

Bile alcohol metabolism in man. Conversion of 5beta-cholestane-3alpha, 7alpha,12alpha, 25-tetrol to cholic acid.

PubMed Central

Salen, G; Shefer, S; Setoguchi, T; Mosbach, E H

1975-01-01

To study the role of C25-HYDROXY BILE ALCOHOLS AS PRECURSORS OF CHOlic acid, [G-3-H]5beta-cholestane-3alpha,7alpha12alpha,25-tetrol was administered intravenously to two subjects with cerebrotendinous xanthomatosis (CTX) and two normal individuals. One day after pulse labeling, radioactivity was present in the cholic acid isolated from the bile and feces of the subjects with CTX and the bile of the normal individuals. In the two normal subjects, the sp act decay curves of [G-3-H]-cholic acid were exponential, and no traces of [G-3-H]-5beta-cholestane-3alpha,7alpha,12alpha,25-tetrol were detected. In contrast, appreciable quantities of labeled 5beta-cholestane-3alpha,-7aopha,12alpha,25-tetrol were present in the bile and feces of the CTX subjects. The sp act vs. time curves of fecal [G-3-H]5beta-cholestane-3alpha,7alpha,12alpha,25-tetrol and [G-3-H]-cholic acid showed a precursor-product relationship. Although these results suggest that 5beta-cholestane-3alpha,7alpha,12alpha,25-tetrol may be a precursor of cholic acid in man, the possibility that C26-hydroxy intermediates represent the normal pathway can not be excluded. PMID:1141434

A yeast mutant defective at an early stage in import of secretory protein precursors into the endoplasmic reticulum

PubMed Central

1987-01-01

We have devised a genetic selection for mutant yeast cells that fail to translocate secretory protein precursors into the lumen of the endoplasmic reticulum (ER). Mutant cells are selected by a procedure that requires a signal peptide-containing cytoplasmic enzyme chimera to remain in contact with the cytosol. This approach has uncovered a new secretory mutant, sec61, that is thermosensitive for growth and that accumulates multiple secretory and vacuolar precursor proteins that have not acquired any detectable posttranslational modifications associated with translocation into the ER. Preproteins that accumulate at the sec61 block sediment with the particulate fraction, but are exposed to the cytosol as judged by sensitivity to proteinase K. Thus, the sec61 mutation defines a gene that is required for an early cytoplasmic or ER membrane-associated step in protein translocation. PMID:3305520

Tumour Necrosis Factor-alpha and Nuclear Factor-kappa B Gene Variants in Sepsis.

PubMed

Acar, Leyla; Atalan, Nazan; Karagedik, E Hande; Ergen, Arzu

2018-01-20

The humoral system is activated and various cytokines are released due to infections in tissues and traumatic damage. Nuclear factor-kappa B dimers are encoded by nuclear factor-kappa B genes and regulate transcription of several crucial proteins of inflammation such as tumour necrosis factor-alpha. To investigate the possible effect of polymorphisms on tumour necrosis factor-alpha serum levels with clinical and prognostic parameters of sepsis by determining the nuclear factor-kappa B-1-94 ins/del ATTG and tumour necrosis factor-alpha (-308 G/A) gene polymorphisms and tumour necrosis factor-alpha serum levels. Case-control study. Seventy-two patients with sepsis and 104 healthy controls were included in the study. In order to determine the polymorphisms of nuclear factor-kappa B-1-94 ins/del ATTG and tumour necrosis factor-alpha (-308 G/A), polymerase chain reaction-restriction fragment length polymorphism analysis was performed and serum tumour necrosis factor-alpha levels were determined using an enzyme-linked immunosorbent assay. We observed no significant differences in tumour necrosis factor-alpha serum levels between the study groups. In the patient group, an increase in the tumour necrosis factor-alpha serum levels in patients carrying the tumour necrosis factor-alpha (-308 G/A) A allele compared to those without the A allele was found to be statistically significant. Additionally, an increase in the tumour necrosis factor-alpha serum levels in patients carrying tumour necrosis factor-alpha (-308 G/A) AA genotype compared with patients carrying the AG or GG genotypes was statistically significant. No significant differences were found in these 2 polymorphisms between the patient and control groups (p>0.05). Our results showed the AA genotype and the A allele of the tumour necrosis factor-alpha (-308 G/A) polymorphism may be used as a predictor of elevated tumour necrosis factor-alpha levels in patients with sepsis.

Production of the forskolin precursor 11β-hydroxy-manoyl oxide in yeast using surrogate enzymatic activities.

PubMed

Ignea, Codruta; Ioannou, Efstathia; Georgantea, Panagiota; Trikka, Fotini A; Athanasakoglou, Anastasia; Loupassaki, Sofia; Roussis, Vassilios; Makris, Antonios M; Kampranis, Sotirios C

2016-02-26

Several plant diterpenes have important biological properties. Among them, forskolin is a complex labdane-type diterpene whose biological activity stems from its ability to activate adenylyl cyclase and to elevate intracellular cAMP levels. As such, it is used in the control of blood pressure, in the protection from congestive heart failure, and in weight-loss supplements. Chemical synthesis of forskolin is challenging, and production of forskolin in engineered microbes could provide a sustainable source. To this end, we set out to establish a platform for the production of forskolin and related epoxy-labdanes in yeast. Since the forskolin biosynthetic pathway has only been partially elucidated, and enzymes involved in terpene biosynthesis frequently exhibit relaxed substrate specificity, we explored the possibility of reconstructing missing steps of this pathway employing surrogate enzymes. Using CYP76AH24, a Salvia pomifera cytochrome P450 responsible for the oxidation of C-12 and C-11 of the abietane skeleton en route to carnosic acid, we were able to produce the forskolin precursor 11β-hydroxy-manoyl oxide in yeast. To improve 11β-hydroxy-manoyl oxide production, we undertook a chassis engineering effort involving the combination of three heterozygous yeast gene deletions (mct1/MCT1, whi2/WHI2, gdh1/GDH1) and obtained a 9.5-fold increase in 11β-hydroxy-manoyl oxide titers, reaching 21.2 mg L(-1). In this study, we identify a surrogate enzyme for the specific and efficient hydroxylation of manoyl oxide at position C-11β and establish a platform that will facilitate the synthesis of a broad range of tricyclic (8,13)-epoxy-labdanes in yeast. This platform forms a basis for the heterologous production of forskolin and will facilitate the elucidation of subsequent steps of forskolin biosynthesis. In addition, this study highlights the usefulness of using surrogate enzymes for the production of intermediates of complex biosynthetic pathways. The combination of

[Building immune microsphere against tumor necrosis factor-alpha (TNF-alpha)].

PubMed

Wang, Qin; Wu, Xiongfei; Wang, Junxia; Liu, Hong; Li, Lian; Jin, Xiyu

2005-12-01

We have constructed the immune microsphere against tumor necrosis factor-alpha (TNF-alpha) prospectively, hoping to establish the experiment groundwork in more researches which could be used in specific elimination of the TNF-alpha by blood purification method for the future. The recombinant human tumor necrosis factor-alpha monoclonal antibody (rHTNF-alpha McAb) was wrapped on the polystyrene microsphere (PSM) carrier connecting poly-L-lysine (PLL) beforehand. They were earmarked by the fluorescein isothiocyanate (FITC) respectively. The packing conditions were examined using the inversted and fluorescence microscopes and the spectrophotometer. The results showed that the best conditions for wrapping were 20 degrees C, pH9.5 and 60 minutes. The PLL content was not changed in the washing fluid after coating, which indicated the wrapping was quite firm. At the same temperature and same coating time, the rHTNF-alpha McAb coated on the PLL was obviously substantial when the concentration of glutaraldehyde solution was 0.2%. The findings demonstrated that the built immune microsphers can be used as a novel adsorption material. This method is simple and economic, and it offers a new approach to the related studies.

Recovery of Fuel-Precursor Lipids from Oleaginous Yeast

DOE PAGES

Kruger, Jacob S.; Cleveland, Nicholas S.; Yeap, Rou Yi; ...

2018-01-24

Bio-derived lipids offer a potentially promising intermediate to displace petroleum-derived diesel. One of the key challenges for the production of lipids via microbial cell mass is that these products are stored intracellularly and must be extracted and recovered efficiently and economically. Thus, improved methods of cell lysis and lipid extraction are needed. In this study, we examine lipid extraction from wet oleaginous yeast in combination with seven different cell lysis approaches encompassing both physical and chemical techniques (high-pressure homogenization, microwave and conventional thermal treatments, bead beating, acid, base, and enzymatic treatments) to facilitate lipid extraction from a model oleaginous yeastmore » strain, Lipomyces starkeyi. Of the seven techniques investigated, acid treatment led to the highest lipid recovery yields. Further exploration of acid treatment and integration with an economic model revealed that treatment at 170 degrees C for 60 min at 1 wt% H 2SO 4 and 8 wt% yeast solids represents a viable option for both lipid recovery yield and process economics, enabling experimental lipid recovery yields of 88.5-93.0% to be achieved at a corresponding estimated minimum fuel selling price (MFSP) of $5.13-$5.61/gallon of gasoline equivalent (GGE). The same acid treatment conditions applied to two other strains of oleaginous yeast (Cutaneotrichosporon curvatus and Rhodotorula toruloides) resulted in similar lipid recovery yields. In pretreatment experiments scaled up to 300 mL, slightly lower temperatures or shorter pretreatment times, along with higher yeast solids loading, resulted in higher lipid yields than the conditions identified from the small-scale runs. Two replicate runs carried out at 170 degrees C for 30 min using 1 wt% H2SO4 and 19 wt% yeast solids achieved an average lipid recovery of 96.1% at a corresponding estimated MFSP of $4.89/GGE. In all cases, the lipids are primarily triglycerides and free fatty acids

Recovery of Fuel-Precursor Lipids from Oleaginous Yeast

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kruger, Jacob S.; Cleveland, Nicholas S.; Yeap, Rou Yi

Bio-derived lipids offer a potentially promising intermediate to displace petroleum-derived diesel. One of the key challenges for the production of lipids via microbial cell mass is that these products are stored intracellularly and must be extracted and recovered efficiently and economically. Thus, improved methods of cell lysis and lipid extraction are needed. In this study, we examine lipid extraction from wet oleaginous yeast in combination with seven different cell lysis approaches encompassing both physical and chemical techniques (high-pressure homogenization, microwave and conventional thermal treatments, bead beating, acid, base, and enzymatic treatments) to facilitate lipid extraction from a model oleaginous yeastmore » strain, Lipomyces starkeyi. Of the seven techniques investigated, acid treatment led to the highest lipid recovery yields. Further exploration of acid treatment and integration with an economic model revealed that treatment at 170 degrees C for 60 min at 1 wt% H 2SO 4 and 8 wt% yeast solids represents a viable option for both lipid recovery yield and process economics, enabling experimental lipid recovery yields of 88.5-93.0% to be achieved at a corresponding estimated minimum fuel selling price (MFSP) of $5.13-$5.61/gallon of gasoline equivalent (GGE). The same acid treatment conditions applied to two other strains of oleaginous yeast (Cutaneotrichosporon curvatus and Rhodotorula toruloides) resulted in similar lipid recovery yields. In pretreatment experiments scaled up to 300 mL, slightly lower temperatures or shorter pretreatment times, along with higher yeast solids loading, resulted in higher lipid yields than the conditions identified from the small-scale runs. Two replicate runs carried out at 170 degrees C for 30 min using 1 wt% H2SO4 and 19 wt% yeast solids achieved an average lipid recovery of 96.1% at a corresponding estimated MFSP of $4.89/GGE. In all cases, the lipids are primarily triglycerides and free fatty acids

Stereoselective Synthesis of [alpha, alpha][superscript ']-Biprolines

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vartak, Ashish P.; Young, Jr., Victor G.; Johnson, Rodney L.

2010-11-10

A means to induce dehydrodimerization of Seebach's oxazolidinone (5), the stereochemical outcome of which is entirely temperature dependent, is described. The resultant dimers 3 and 4 are precursors to (R,R)-alpha,alpha'-biproline (1) and meso-alpha,alpha'-biproline (2), respectively. An organohypobromite and an iminium halide are proposed to serve as electrophiles in the reaction with the enolate of 5 to give 3 and 4, respectively.

Alpha-ketoglutarate enhances freeze-thaw tolerance and prevents carbohydrate-induced cell death of the yeast Saccharomyces cerevisiae.

PubMed

Bayliak, Maria M; Hrynkiv, Olha V; Knyhynytska, Roksolana V; Lushchak, Volodymyr I

2018-01-01

Stress resistance and fermentative capability are important quality characteristics of baker's yeast. In the present study, we examined protective effects of exogenous alpha-ketoglutarate (AKG), an intermediate of the tricarboxylic acid cycle and amino acid metabolism, against freeze-thaw and carbohydrate-induced stresses in the yeast Saccharomyces cerevisiae. Growth on AKG-supplemented medium prevented a loss of viability and improved fermentative capacity of yeast cells after freeze-thaw treatment. The cells grown in the presence of AKG had higher levels of amino acids (e.g., proline), higher metabolic activity and total antioxidant capacity, and higher activities of catalase, NADP-dependent glutamate dehydrogenase and glutamine synthase compared to control ones. Both synthesis of amino acids and enhancement of antioxidant system capacity could be involved in AKG-improved freeze-thaw tolerance in S. cerevisiae. Cell viability dramatically decreased under incubation of stationary-phase yeast cells in 2% glucose or fructose solutions (in the absence of the other nutrients) as compared with incubation in distilled water or in 10 mM AKG solution. The decrease in cell viability was accompanied by acidification of the medium, and decrease in cellular respiration, aconitase activity, and levels of total protein and free amino acids. The supplementation with 10 mM AKG effectively prevented carbohydrate-induced yeast death. Protective mechanisms of AKG could be associated with the intensification of respiration and prevention of decreasing protein level as well as with direct antioxidant AKG action.

Glycoprotein synthesis in yeast. Identification of Man8GlcNAc2 as an essential intermediate in oligosaccharide processing.

PubMed

Byrd, J C; Tarentino, A L; Maley, F; Atkinson, P H; Trimble, R B

1982-12-25

Synthesis of the N-linked oligosaccharides of Saccharomyces cerevisiae glycoproteins has been studied in vivo by labeling with [2-3H]mannose and gel filtration analysis of the products released by endoglycosidase H. Both small oligosaccharides, Man8-14GlcNAc, and larger products, Man greater than 20GlcNAc, were labeled. The kinetics of continuous and pulse-chase labeling demonstrated that Glc3Man9GlcNAc2, the initial product transferred to protein, was rapidly (t1/2 congruent to 3 min) trimmed to Man8GlcNAc2 and then more slowly (t1/2 = 10-20 min) elongated to larger oligosaccharides. No oligosaccharides smaller than Man8GlcNAc2 were evident with either labeling procedure. In confirmation of the trimming reaction observed in vivo, 3H-labeled Man9-N-acetylglucosaminitol from bovine thyroglobulin and [14C]Man9GlcNAc2 from yeast oligosaccharide-lipid were converted in vitro by broken yeast cells to 3H-labeled Man8-N-acetylglucosaminitol and [14C]Man8GlcNAc2. Man8GlcNAc and Man9GlcNAc from yeast invertase and from bovine thyroglobulin were purified by gel filtration and examined by high field 1H-NMR analysis. Invertase Man8GlcNAc (B) and Man9GlcNAc (C) were homogeneous compounds, which differed from the Man9GlcNAc (A) of thyroglobulin by the absence of a specific terminal alpha 1,2-linked mannose residue. The Man9GlcNAc of invertase (C) had an additional terminal alpha 1,6-linked mannose and appeared identical in structure with that isolated from yeast containing the mnn1 and mnn2 mutations (Cohen, R. E., Zhang, W.-j., and Ballou, C. E. (1982) J. Biol. Chem. 257, 5730-5737). It is concluded that Man8GlcNAc2, formed by removal of glucose and a single mannose from Glc3Man9GlcNAc2, is the ultimate product of trimming and the minimal precursor for elongation of the oligosaccharides on yeast glycoproteins. The results suggest that removal of a particular terminal alpha 1,2-linked mannose from Man9GlcNAc2 by a highly specific alpha-mannosidase exposes the nascent Man-alpha 1

The development of bactericidal yeast strains by expressing the Pediococcus acidilactici pediocin gene (pedA) in Saccharomyces cerevisiae.

PubMed

Schoeman, H; Vivier, M A; Du Toit, M; Dicks, L M; Pretorius, I S

1999-06-15

The excessive use of sulphur dioxide and other chemical preservatives in wine, beer and other fermented food and beverage products to prevent the growth of unwanted microbes holds various disadvantages for the quality of the end-products and is confronted by mounting consumer resistance. The objective of this study was to investigate the feasibility of controlling spoilage bacteria during yeast-based fermentations by engineering bactericidal strains of Saccharomyces cerevisiae. To test this novel concept, we have successfully expressed a bacteriocin gene in yeast. The pediocin operon of Pediococcus acidilactici PAC1.0 consists of four clustered genes, namely pedA (encoding a 62 amino acid precursor of the PA-1 pediocin), pedB (encoding an immunity factor), pedC (encoding a PA-1 transport protein) and pedD (encoding a protein involved in the transport and processing of PA-1). The pedA gene was inserted into a yeast expression/secretion cassette and introduced as a multicopy episomal plasmid into a laboratory strain (Y294) of S. cerevisiae. Northern blot analysis confirmed that the pedA structural gene in this construct (ADH1P-MFa1S-pedA-ADH1T, designated PED1), was efficiently expressed under the control of the yeast alcohol dehydrogenase I gene promoter (ADH1P) and terminator (ADH1T). Secretion of the PED1-encoded pediocin PA-1 was directed by the yeast mating pheromone alpha-factor's secretion signal (MFa1S). The presence of biologically active antimicrobial peptides produced by the yeast transformants was indicated by agar diffusion assays against sensitive indicator bacteria (e.g. Listeria monocytogenes B73). Protein analysis indicated the secreted heterologous peptide to be approximately 4.6 kDa, which conforms to the expected size. The heterologous peptide was present at relatively low levels in the yeast supernatant but pediocin activity was readily detected when intact yeast colonies were used in sensitive strain overlays. This study could lead to the

The alpha subunit of the Saccharomyces cerevisiae oligosaccharyltransferase complex is essential for vegetative growth of yeast and is homologous to mammalian ribophorin I

PubMed Central

1995-01-01

Oligosaccharyltransferase mediates the transfer of a preassembled high mannose oligosaccharide from a lipid-linked oligosaccharide donor to consensus glycosylation acceptor sites in newly synthesized proteins in the lumen of the rough endoplasmic reticulum. The Saccharomyces cerevisiae oligosaccharyltransferase is an oligomeric complex composed of six nonidentical subunits (alpha-zeta), two of which are glycoproteins (alpha and beta). The beta and delta subunits of the oligosaccharyltransferase are encoded by the WBP1 and SWP1 genes. Here we describe the functional characterization of the OST1 gene that encodes the alpha subunit of the oligosaccharyltransferase. Protein sequence analysis revealed a significant sequence identity between the Saccharomyces cerevisiae Ost1 protein and ribophorin I, a previously identified subunit of the mammalian oligosaccharyltransferase. A disruption of the OST1 locus was not tolerated in haploid yeast showing that expression of the Ost1 protein is essential for vegetative growth of yeast. An analysis of a series of conditional ost1 mutants demonstrated that defects in the Ost1 protein cause pleiotropic underglycosylation of soluble and membrane-bound glycoproteins at both the permissive and restrictive growth temperatures. Microsomal membranes isolated from ost1 mutant yeast showed marked reductions in the in vitro transfer of high mannose oligosaccharide from exogenous lipid-linked oligosaccharide to a glycosylation site acceptor tripeptide. Microsomal membranes isolated from the ost1 mutants contained elevated amounts of the Kar2 stress-response protein. PMID:7860628

The noradrenaline precursor L-threo-3,4-dihydroxyphenylserine exhibits antinociceptive activity via central alpha-adrenoceptors in the mouse.

PubMed Central

Kawabata, A.; Kasamatsu, K.; Umeda, N.; Takagi, H.

1994-01-01

1. Systemic (s.c. or p.o.) administration of L-threo-3,4-dihydroxyphenylserine (droxidopa, L-threo-DOPS; L-DOPS), a noradrenaline precursor, at a dose-range of 100-800 mg kg-1, produced naloxone-resistant antinociception in a dose-dependent manner in the mouse, as assessed by the tail flick test, kaolin-induced writhing test and formalin-induced nociception test. 2. Antinociception elicited by L-DOPS (400 mg kg-1, s.c.) was not affected by s.c. injection of benserazide, a peripherally preferential L-aromatic amino acid decarboxylase inhibitor, but was suppressed by its intracerebroventricular (i.c.v.) injection. 3. I.c.v. or intrathecal (i.t.) administration of the non-selective alpha-blocker, phentolamine, significantly reduced L-DOPS-induced antinociception. 4. I.c.v. administration of the alpha 1-blocker, prazosin, but not the alpha 2-blocker, yohimbine, abolished the antinociceptive effects of L-DOPS. In contrast, both blockers, when administered i.t., exhibited significant inhibitory effects. 5. These results suggest that systemic L-DOPS produces opioid-independent antinociception, mediated by supraspinal alpha 1-adrenoceptors and by spinal alpha 1- and alpha 2-adrenoceptors and may predict additional therapeutic applications of L-DOPS as an analgesic. PMID:7911717

Increased plasma orexin-A levels in patients with insomnia disorder are not associated with prepro-orexin or orexin receptor gene polymorphisms.

PubMed

Tang, Shi; Huang, Weiwei; Lu, Shanshan; Lu, Lili; Li, Guohua; Chen, Xu; Liu, Xiaomin; Lv, Xin; Zhao, Zhangning; Duan, Ruisheng; Du, Yifeng; Tang, Jiyou

2017-02-01

Orexins, also known as hypocretins, play a regulatory role in the sleep-wake cycle by activating orexin receptors. Previous animal studies have shown that sleep deprivation can elevate orexinergic peptide levels. However, the relationship between insomnia disorder and orexin-A levels in humans has not been explored. In the current study, we examined plasma orexin-A levels in patients with insomnia disorder and in normal sleepers. We also studied the possible mechanisms underlying changes in orexin-A levels between the study groups; this included investigations of prepro-orexin and orexin receptor gene polymorphisms as well as exploration of other variables. We measured plasma orexin-A levels in 228 patients with insomnia disorder and 282 normal sleepers. The results indicated that the patients with insomnia disorder had significantly higher orexin-A levels than normal sleepers (63.42±37.56 vs. 54.84±23.95pg/ml). A positive relationship was detected between orexin-A level and age in patients with insomnia disorder. Orexin-A levels were elevated in relation to course of insomnia, as well as in relation to increased Insomnia Severity Index score. None of the evaluated prepro-orexin gene single nucleotide polymorphisms were informative between the two study populations. After sequencing all orexin receptor exons, one variation (rs2271933) in the OX1R gene and one variation (rs2653349) in the OX2R gene were found. However, no significant differences were found in either genotypic or allelic frequency distributions between the two study groups. It is suggested that the increased plasma orexin-A levels in patients with insomnia disorder are associated with the course and severity of insomnia, but not with prepro-orexin and orexin receptor gene polymorphisms. Copyright © 2016 Elsevier Inc. All rights reserved.

Air pollution alters brain and pituitary endothelin-1 and inducible nitric oxide synthase gene expression.

PubMed

Thomson, Errol M; Kumarathasan, Prem; Calderón-Garcidueñas, Lilian; Vincent, Renaud

2007-10-01

Recent work suggests that air pollution is a risk factor for cerebrovascular and neurodegenerative disease. Effects of inhaled pollutants on the production of vasoactive factors such as endothelin (ET) and nitric oxide (NO) in the brain may be relevant to disease pathogenesis. Inhaled pollutants increase circulating levels of ET-1 and ET-3, and the pituitary is a potential source of plasma ET, but the effects of pollutants on the expression of ET and NO synthase genes in the brain and pituitary are not known. In the present study, Fischer-344 rats were exposed by nose-only inhalation to particles (0, 5, 50mg/m3 EHC-93), ozone (0, 0.4, 0.8 ppm), or combinations of particles and ozone for 4 h. Real-time reverse transcription polymerase chain reaction was used to measure mRNA levels in the cerebral hemisphere and pituitary 0 and 24 h post-exposure. Ozone inhalation significantly increased preproET-1 but decreased preproET-3 mRNAs in the cerebral hemisphere, while increasing mRNA levels of preproET-1, preproET-3, and the ET-converting enzyme (ECE)-1 in the pituitary. Inducible NO synthase (iNOS) was initially decreased in the cerebral hemisphere after ozone inhalation, but increased 24 h post-exposure. Particles decreased tumour necrosis factor (TNF)-alpha mRNA in the cerebral hemisphere, and both particles and ozone decreased TNF-alpha mRNA in the pituitary. Our results show that ozone and particulate matter rapidly modulate the expression of genes involved in key vasoregulatory pathways in the brain and pituitary, substantiating the notion that inhaled pollutants induce cerebrovascular effects.

Alpha Power Modulates Perception Independently of Endogenous Factors.

PubMed

Brüers, Sasskia; VanRullen, Rufin

2018-01-01

Oscillations are ubiquitous in the brain. Alpha oscillations in particular have been proposed to play an important role in sensory perception. Past studies have shown that the power of ongoing EEG oscillations in the alpha band is negatively correlated with visual outcome. Moreover, it also co-varies with other endogenous factors such as attention, vigilance, or alertness. In turn, these endogenous factors influence visual perception. Therefore, it remains unclear how much of the relation between alpha and perception is indirectly mediated by such endogenous factors, and how much reflects a direct causal influence of alpha rhythms on sensory neural processing. We propose to disentangle the direct from the indirect causal routes by introducing modulations of alpha power, independently of any fluctuations in endogenous factors. To this end, we use white-noise sequences to constrain the brain activity of 20 participants. The cross-correlation between the white-noise sequences and the concurrently recorded EEG reveals the impulse response function (IRF), a model of the systematic relationship between stimulation and brain response. These IRFs are then used to reconstruct rather than record the brain activity linked with new random sequences (by convolution). Interestingly, this reconstructed EEG only contains information about oscillations directly linked to the white-noise stimulation; fluctuations in attention and other endogenous factors may still modulate brain alpha rhythms during the task, but our reconstructed EEG is immune to these factors. We found that the detection of near-perceptual threshold targets embedded within these new white-noise sequences depended on the power of the ~10 Hz reconstructed EEG over parieto-occipital channels. Around the time of presentation, higher power led to poorer performance. Thus, fluctuations in alpha power, induced here by random luminance sequences, can directly influence perception: the relation between alpha power and

Induction of multiple pleiotropic drug resistance genes in yeast engineered to produce an increased level of anti-malarial drug precursor, artemisinic acid.

PubMed

Ro, Dae-Kyun; Ouellet, Mario; Paradise, Eric M; Burd, Helcio; Eng, Diana; Paddon, Chris J; Newman, Jack D; Keasling, Jay D

2008-11-04

Due to the global occurrence of multi-drug-resistant malarial parasites (Plasmodium falciparum), the anti-malarial drug most effective against malaria is artemisinin, a natural product (sesquiterpene lactone endoperoxide) extracted from sweet wormwood (Artemisia annua). However, artemisinin is in short supply and unaffordable to most malaria patients. Artemisinin can be semi-synthesized from its precursor artemisinic acid, which can be synthesized from simple sugars using microorganisms genetically engineered with genes from A. annua. In order to develop an industrially competent yeast strain, detailed analyses of microbial physiology and development of gene expression strategies are required. Three plant genes coding for amorphadiene synthase, amorphadiene oxidase (AMO or CYP71AV1), and cytochrome P450 reductase, which in concert divert carbon flux from farnesyl diphosphate to artemisinic acid, were expressed from a single plasmid. The artemisinic acid production in the engineered yeast reached 250 microg mL(-1) in shake-flask cultures and 1 g L(-1) in bio-reactors with the use of Leu2d selection marker and appropriate medium formulation. When plasmid stability was measured, the yeast strain synthesizing amorphadiene alone maintained the plasmid in 84% of the cells, whereas the yeast strain synthesizing artemisinic acid showed poor plasmid stability. Inactivation of AMO by a point-mutation restored the high plasmid stability, indicating that the low plasmid stability is not caused by production of the AMO protein but by artemisinic acid synthesis or accumulation. Semi-quantitative reverse-transcriptase (RT)-PCR and quantitative real time-PCR consistently showed that pleiotropic drug resistance (PDR) genes, belonging to the family of ATP-Binding Cassette (ABC) transporter, were massively induced in the yeast strain producing artemisinic acid, relative to the yeast strain producing the hydrocarbon amorphadiene alone. Global transcriptional analysis by yeast

Combined Expression of Aspergillus nidulans Endoxylanase X24 and Aspergillus oryzae (alpha)-Amylase in Industrial Baker's Yeasts and Their Use in Bread Making.

PubMed

Monfort, A; Blasco, A; Prieto, J A; Sanz, P

1996-10-01

The Aspergillus nidulans endoxylanase X24 and the Aspergillus oryzae (alpha)-amylase cDNAs were placed under the control of the Saccharomyces cerevisiae actin promoter (pACT1) and introduced into baker's yeast. Bread made with transformants expressing both enzymes (YEpACT-AMY-ACT-X24) showed a 30% increase in volume and reduced firmness in comparison with that produced with a commercial strain. Endoxylanase X24 and (alpha)-amylase seem to act synergistically to improve the quality of bread in terms of volume and density.

Sphingosine mediates the immediate negative inotropic effects of tumor necrosis factor-alpha in the adult mammalian cardiac myocyte.

PubMed

Oral, H; Dorn, G W; Mann, D L

1997-02-21

To determine whether activation of the neutral sphingomyelinase pathway was responsible for the immediate (<30 min) negative inotropic effects of tumor necrosis factor-alpha (TNF-alpha), we examined sphingosine levels in diluent and TNF-alpha-stimulated cardiac myocytes. TNF-alpha stimulation of adult feline cardiac myocytes provoked a rapid (<15 min) increase in the hydrolysis of [14C]sphingomyelin in cell-free extracts, as well as an increase in ceramide mass, consistent with cytokine-induced activation of the neutral sphingomyelinase pathway. High performance liquid chromatographic analysis of lipid extracts from TNF-alpha-stimulated cardiac myocytes showed that TNF-alpha stimulation produced a rapid (<30 min) increase in free sphingosine levels. Moreover, exogenous D-sphingosine mimicked the effects of TNF-alpha on intracellular calcium homeostasis, as well as the negative inotropic effects of TNF-alpha in isolated contracting myocytes; time course studies showed that exogenous D-sphingosine produced abnormalities in cell shortening that were maximal at 5 min. Finally, blocking sphingosine production using an inhibitor of ceramidase, n-oleoylethanolamine, completely abrogated the negative inotropic effects of TNF-alpha in isolated contracting cardiac myocytes. Additional studies employing biologically active ceramide analogs and sphingosine 1-phosphate suggested that neither the immediate precursor of sphingosine nor the immediate metabolite of sphingosine, respectively, were likely to be responsible for the immediate negative inotropic effects of TNF-alpha. Thus, these studies suggest that sphingosine mediates the immediate negative inotropic effects of TNF-alpha in isolated cardiac myocytes.

Negative interference by rheumatoid factor in alpha-fetoprotein chemiluminescent microparticle immunoassay.

PubMed

Wang, Hui; Bi, Xiaohui; Xu, Lei; Li, Yirong

2017-01-01

Background Rheumatoid factor causes positive interference in multiple immunoassays. Recently, negative interference has also been found in immunoassays in the presence of rheumatoid factor. The chemiluminescent microparticle immunoassay is widely used to determine serum alpha-fetoprotein. However, it is not clear whether the presence of rheumatoid factor in the serum causes interference in the chemiluminescent microparticle immunoassay of alpha-fetoprotein. Methods Serum alpha-fetoprotein was determined using the ARCHITECT alpha-fetoprotein assay. The estimation of alpha-fetoprotein recovery was carried out in samples prepared by diluting high-concentration alpha-fetoprotein serum with rheumatoid factor-positive or rheumatoid factor-negative serum. Paramagnetic microparticles coated with hepatitis B surface antigen-anti-HBs complexes were used to remove rheumatoid factor from the serum. Results The average recovery of alpha-fetoprotein was 88.4% and 93.8% in the rheumatoid factor-positive and rheumatoid factor-negative serum samples, respectively. The recovery of alpha-fetoprotein was significantly lower in the rheumatoid factor-positive serum samples than in the rheumatoid factor-negative serum samples. In two of five rheumatoid factor-positive samples, a large difference was found (9.8%) between the average alpha-fetoprotein recoveries in the serially diluted and initial recoveries. Fourteen rheumatoid factor-positive serum samples were pretreated with hepatitis B surface antigen-anti-HBs complex-coated paramagnetic microparticles. The alpha-fetoprotein concentrations measured in the pretreated samples increased significantly. Conclusions It was concluded that the alpha-fetoprotein chemiluminescent microparticle immunoassay is susceptible to interference by rheumatoid factor, leading to significantly lower results. Eliminating the incidence of negative interference from rheumatoid factor should be an important goal for immunoassay providers. In the meantime

Combined Expression of Aspergillus nidulans Endoxylanase X24 and Aspergillus oryzae (alpha)-Amylase in Industrial Baker's Yeasts and Their Use in Bread Making

PubMed Central

Monfort, A.; Blasco, A.; Prieto, J. A.; Sanz, P.

1996-01-01

The Aspergillus nidulans endoxylanase X24 and the Aspergillus oryzae (alpha)-amylase cDNAs were placed under the control of the Saccharomyces cerevisiae actin promoter (pACT1) and introduced into baker's yeast. Bread made with transformants expressing both enzymes (YEpACT-AMY-ACT-X24) showed a 30% increase in volume and reduced firmness in comparison with that produced with a commercial strain. Endoxylanase X24 and (alpha)-amylase seem to act synergistically to improve the quality of bread in terms of volume and density. PMID:16535419

Interaction of plant chimeric calcium/calmodulin-dependent protein kinase with a homolog of eukaryotic elongation factor-1alpha

NASA Technical Reports Server (NTRS)

Wang, W.; Poovaiah, B. W.

1999-01-01

A chimeric Ca2+/calmodulin-dependent protein kinase (CCaMK) was previously cloned and characterized in this laboratory. To investigate the biological functions of CCaMK, the yeast two-hybrid system was used to isolate genes encoding proteins that interact with CCaMK. One of the cDNA clones obtained from the screening (LlEF-1alpha1) has high similarity with the eukaryotic elongation factor-1alpha (EF-1alpha). CCaMK phosphorylated LlEF-1alpha1 in a Ca2+/calmodulin-dependent manner. The phosphorylation site for CCaMK (Thr-257) was identified by site-directed mutagenesis. Interestingly, Thr-257 is located in the putative tRNA-binding region of LlEF-1alpha1. An isoform of Ca2+-dependent protein kinase (CDPK) phosphorylated multiple sites of LlEF-1alpha1 in a Ca2+-dependent but calmodulin-independent manner. Unlike CDPK, CCaMK phosphorylated only one site, and this site is different from CDPK phosphorylation sites. This suggests that the phosphorylation of EF-1alpha by these two kinases may have different functional significance. Although the phosphorylation of LlEF-1alpha1 by CCaMK is Ca2+/calmodulin-dependent, in vitro binding assays revealed that CCaMK binds to LlEF-1alpha1 in a Ca2+-independent manner. This was further substantiated by coimmunoprecipitation of CCaMK and EF-1alpha using the protein extract from lily anthers. Dissociation of CCaMK from EF-1alpha by Ca2+ and phosphorylation of EF-1alpha by CCaMK in a Ca2+/calmodulin-dependent manner suggests that these interactions may play a role in regulating the biological functions of EF-1alpha.

Control of yeast mating signal transduction by a mammalian. beta. sub 2 -adrenergic receptor and G sub s. alpha. subunit

DOE Office of Scientific and Technical Information (OSTI.GOV)

King, K.; Caron, M.G.; Lefkowitz, R.J.

1990-10-05

To facilitate functional and mechanistic studies of receptor-G protein interactions by expression of the human {beta}{sub 2}-adrenergic receptor (h{beta}-AR) has been expressed in Saccharomyces cerevisiae. This was achieved by placing a modified h{beta}-AR gene under control of the galactose-inducible GAL1 promoter. After induction by galactose, functional h{beta}-AR was expressed at a concentration several hundred times as great as that found in any human tissue. As determined from competitive ligand binding experiments, h{beta}-AR expressed in yeast displayed characteristic affinities, specificity, and stereoselectivity. Partial activation of the yeast pheromone response pathway by {beta}-adrenergic receptor agonists was achieved in cells coexpressing h{beta}-AR andmore » a mammalian G protein (G{sub s}) {alpha} subunit - demonstrating that these components can couple to each other and to downstream effectors when expressed in yeast. This in vivo reconstitution system provides a new approach for examining ligand binding and G protein coupling to cell surface receptors.« less

Tumor necrosis factor-{alpha} enhances IL-15-induced natural killer cell differentiation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Jiwon; Lee, Suk Hyung; Korea University of Science and Technology, Yusong, Daejeon 305-333

2009-09-04

The differentiation of natural killer (NK) cells is regulated by various factors including soluble growth factors and transcription factors. Here, we have demonstrated that tumor necrosis factor-{alpha} (TNF-{alpha}) is a positive regulator of NK cell differentiation. TNF-{alpha} augmented the IL-15-induced expression of NK1.1 and CD122 in mature NK cells, and TNF-{alpha} alone also induced NK cell maturation as well as IL-15. TNF-{alpha} also increased IFN-{gamma} production in NK cells in the presence of IL-15. Meanwhile, mRNA expression of several transcription factors, including T-bet and GATA-3, was increased by the addition of TNF-{alpha} and IL-15. In addition, TNF-{alpha} increased nuclear factor-kappamore » B (NF-{kappa}B) activity in NK cells and inhibition of NF-{kappa}B impeded TNF-{alpha}-enhanced NK cell maturation. Overall, these data suggest that TNF-{alpha} significantly increased IL-15-driven NK cell differentiation by increasing the expression of transcription factors that play crucial roles in NK cell maturation and inducing the NF-{kappa}B activity.« less

NMR study of the transforming growth factor-alpha (TGF-alpha)-epidermal growth factor receptor complex. Visualization of human TGF-alpha binding determinants through nuclear Overhauser enhancement analysis.

PubMed

McInnes, C; Hoyt, D W; Harkins, R N; Pagila, R N; Debanne, M T; O'Connor-McCourt, M; Sykes, B D

1996-12-13

The study of human transforming growth factor-alpha (TGF-alpha) in complex with the epidermal growth factor (EGF) receptor extracellular domain has been undertaken in order to generate information on the interactions of these molecules. Analysis of 1H NMR transferred nuclear Overhauser enhancement data for titration of the ligand with the receptor has yielded specific data on the residues of the growth factor involved in contact with the larger protein. Significant increases and decreases in nuclear Overhauser enhancement cross-peak intensity occur upon complexation, and interpretation of these changes indicates that residues of the A- and C-loops of TGF-alpha form the major binding interface, while the B-loop provides a structural scaffold for this site. These results corroborate the conclusions from NMR relaxation studies (Hoyt, D. W., Harkins, R. N., Debanne, M. T., O'Connor-McCourt, M., and Sykes, B. D. (1994) Biochemistry 33, 15283-15292), which suggest that the C-terminal residues of the polypeptide are immobilized upon receptor binding, while the N terminus of the molecule retains considerable flexibility, and are consistent with structure-function studies of the TGF-alpha/EGF system indicating a multidomain binding model. These results give a visualization, for the first time, of native TGF-alpha in complex with the EGF receptor and generate a picture of the ligand-binding site based upon the intact molecule. This will undoubtedly be of utility in the structure-based design of TGF-alpha/EGF agonists and/or antagonists.

Storage and computationally efficient permutations of factorized covariance and square-root information matrices

NASA Technical Reports Server (NTRS)

Muellerschoen, R. J.

1988-01-01

A unified method to permute vector-stored upper-triangular diagonal factorized covariance (UD) and vector stored upper-triangular square-root information filter (SRIF) arrays is presented. The method involves cyclical permutation of the rows and columns of the arrays and retriangularization with appropriate square-root-free fast Givens rotations or elementary slow Givens reflections. A minimal amount of computation is performed and only one scratch vector of size N is required, where N is the column dimension of the arrays. To make the method efficient for large SRIF arrays on a virtual memory machine, three additional scratch vectors each of size N are used to avoid expensive paging faults. The method discussed is compared with the methods and routines of Bierman's Estimation Subroutine Library (ESL).

Tumor necrosis factor-alpha-independent downregulation of hepatic cholesterol 7alpha-hydroxylase gene in mice treated with lead nitrate.

PubMed

Kojima, Misaki; Sekikawa, Kenji; Nemoto, Kiyomitsu; Degawa, Masakuni

2005-10-01

We previously reported that lead nitrate (LN), an inducer of hepatic tumor necrosis factor-alpha (TNF-alpha), downregulated gene expression of cholesterol 7alpha-hydroxylase. Herein, to clarify the role of TNF-alpha in LN-induced downregulation of cholesterol 7alpha-hydroxylase, effects of LN on gene expression of hepatic cholesterol 7alpha-hydroxylase (Cyp7a1) in TNF-alpha-knockout (KO) and TNF-alpha-wild-type (WT) mice were comparatively examined. Gene expression of hepatic Cyp7a1 in both WT and KO mice decreased to less than 5% of the corresponding controls at 6-12 h after treatment with LN (100 mumol/kg body weight, iv). Levels of hepatic TNF-alpha protein in either WT or KO mice were below the detection limit, although expression levels of the TNF-alpha gene markedly increased at 6 h in WT mice by LN treatment, but not in KO mice. In contrast, in both WT and KO mice, levels of hepatic IL-1beta protein, which is known to be a suppressor of the cholesterol 7alpha-hydroxylase gene in hamsters, were significantly increased 3-6 h after LN treatment. Furthermore, LN-induced downregulation of the Cyp7a1 gene did not necessarily result from altered gene expression of hepatic transcription factors, including positive regulators (liver X receptor alpha, retinoid X receptor alpha, fetoprotein transcription factor, and hepatocyte nuclear factor 4alpha) and a negative regulator small heterodimer partner responsible for expression of the Cyp7a1 gene. The present findings indicated that LN-induced downregulation of the Cyp7a1 gene in mice did not necessarily occur through a TNF-alpha-dependent pathway and might occur mainly through an IL-1beta-dependent pathway.

Deglycosylation of serum vitamin D3-binding protein by alpha-N-acetylgalactosaminidase detected in the plasma of patients with systemic lupus erythematosus.

PubMed

Yamamoto, N; Naraparaju, V R; Moore, M; Brent, L H

1997-03-01

A serum glycoprotein, Gc protein (vitamin D3-binding protein), can be converted by beta-galactosidase of B cells and sialidase of T cells to a potent macrophage-activating factor (MAF), a protein with N-acetylgalactosamine as the remaining sugar moiety. Thus, Gc protein is the precursor for MAF. Treatment of Gc protein with immobilized beta-galactosidase and sialidase generates a remarkably high titered macrophage-activating factor (GcMAF). When peripheral blood monocytes/ macrophages (designated macrophages) of 33 systemic lupus erythematosus patients were incubated with GcMAF (100 pg/ml), the macrophages of all patients were activated as determined by superoxide generation. However, the precursor activity of patient plasma Gc protein was lost or reduced in these patients. Loss of the precursor activity was the result of deglycosylation of plasma Gc protein by alpha-N-acetylgalactosaminidase activity found in the patient plasma. Levels of plasma alpha-N-acetylgalactosaminidase activity in individual patients had an inverse correlation with the MAF precursor activity of their plasma Gc protein. Deglycosylated Gc protein cannot be converted to macro-phage-activating factor. The resulting defect in macro-phage activation may lead to an inability to clear pathogenic immune complexes. Thus, elevated plasma alpha-N-acetylgalactosaminidase activity resulting in the loss of MAF precursor activity and reduced macro-phage activity may play a role in the pathogenesis of systemic lupus erythematosus.

Quality parameters and RAPD-PCR differentiation of commercial baker's yeast and hybrid strains.

PubMed

El-Fiky, Zaki A; Hassan, Gamal M; Emam, Ahmed M

2012-06-01

Baker's yeast, Saccharomyces cerevisiae, is a key component in bread baking. Total of 12 commercial baker's yeast and 2 hybrid strains were compared using traditional quality parameters. Total of 5 strains with high leavening power and the 2 hybrid strains were selected and evaluated for their alpha-amylase, maltase, glucoamylase enzymes, and compared using random amplified polymorphic DNA (RAPD). The results revealed that all selected yeast strains have a low level of alpha-amylase and a high level of maltase and glucoamylase enzymes. Meanwhile, the Egyptian yeast strain (EY) had the highest content of alpha-amylase and maltase enzymes followed by the hybrid YH strain. The EY and YH strains have the highest content of glucoamylase enzyme almost with the same level. The RAPD banding patterns showed a wide variation among commercial yeast and hybrid strains. The closely related Egyptian yeast strains (EY and AL) demonstrated close similarity of their genotypes. The 2 hybrid strains were clustered to Turkish and European strains in 1 group. The authors conclude that the identification of strains and hybrids using RAPD technique was useful in determining their genetic relationship. These results can be useful not only for the basic research, but also for the quality control in baking factories. © 2012 Institute of Food Technologists®

Genetic and pharmacological suppression of oncogenic mutations in RAS genes of yeast and humans

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schafer, W.R.; Sterne, R.; Thorner, J.

1989-07-28

The activity of an oncoprotein and the secretion of a pheromone can be affected by an unusual protein modification. Specifically, posttranslational modification of yeast-a-factor and Ras protein requires an intermediate of the cholesterol biosynthetic pathway. This modification is apparently essential for biological activity. Studies of yeast mutants blocked in sterol biosynthesis demonstrated that the membrane association and biological activation of the yeast Ras2 protein require mevalonate, a precursor of sterols and other isoprenes such as farnesyl pyrophosphate. Furthermore, drugs that inhibit mevalonate biosynthesis blocked the in vivo action of oncogenic derivatives of human Ras protein in the Xenopus oocyte assay.more » The same drugs and mutations also prevented the posttranslational processing and secretion of yeast a-factor, a peptide that is farnesylated. Thus, the mevalonate requirement for Ras activation may indicate that attachment of a mevalonate-derived (isoprenoid) moiety to Ras proteins is necessary for membrane association and biological function. These observations establish a connection between the cholesterol biosynthetic pathway and transformation by the ras oncogene and offer a novel pharmacological approach to investigating, and possibly controlling, ras-mediated malignant transformations. 50 refs., 3 figs., 3 tabs.« less

Overexpression of human virus surface glycoprotein precursors induces cytosolic unfolded protein response in Saccharomyces cerevisiae

PubMed Central

2011-01-01

Background The expression of human virus surface proteins, as well as other mammalian glycoproteins, is much more efficient in cells of higher eukaryotes rather than yeasts. The limitations to high-level expression of active viral surface glycoproteins in yeast are not well understood. To identify possible bottlenecks we performed a detailed study on overexpression of recombinant mumps hemagglutinin-neuraminidase (MuHN) and measles hemagglutinin (MeH) in yeast Saccharomyces cerevisiae, combining the analysis of recombinant proteins with a proteomic approach. Results Overexpressed recombinant MuHN and MeH proteins were present in large aggregates, were inactive and totally insoluble under native conditions. Moreover, the majority of recombinant protein was found in immature form of non-glycosylated precursors. Fractionation of yeast lysates revealed that the core of viral surface protein aggregates consists of MuHN or MeH disulfide-linked multimers involving eukaryotic translation elongation factor 1A (eEF1A) and is closely associated with small heat shock proteins (sHsps) that can be removed only under denaturing conditions. Complexes of large Hsps seem to be bound to aggregate core peripherally as they can be easily removed at high salt concentrations. Proteomic analysis revealed that the accumulation of unglycosylated viral protein precursors results in specific cytosolic unfolded protein response (UPR-Cyto) in yeast cells, characterized by different action and regulation of small Hsps versus large chaperones of Hsp70, Hsp90 and Hsp110 families. In contrast to most environmental stresses, in the response to synthesis of recombinant MuHN and MeH, only the large Hsps were upregulated whereas sHsps were not. Interestingly, the amount of eEF1A was also increased during this stress response. Conclusions Inefficient translocation of MuHN and MeH precursors through ER membrane is a bottleneck for high-level expression in yeast. Overexpression of these recombinant

The pituitary hormones arginine vasopressin-neurophysin II and oxytocin-neurophysin I show close linkage with interleukin-1 on mouse chromosome 2

DOE Office of Scientific and Technical Information (OSTI.GOV)

Marini, J.C.; Nelson, K.K.; Siracusa, L.D.

1993-01-01

Arginine vasopressin (AVP) and oxytocin (OXT) are posterior pituitary hormones. AVP is involved in fluid homeostasis, while OXT is involved in lactation and parturition. AVP is derived from a larger precursor, prepro-arginine-vasopressin-neurophysin II (prepro-AVP-NP II; AVP), and is physically linked to prepro-oxytocin-neurophysin I (prepro-OXT-NPI1; OXT). The genes for AVP and OXT are separated by only 12 kb of DNA in humans, whereas in the mouse 3.5 kb of intergenic sequence lies between Avp and Oxt. Interspecific backcross analysis has now been used to map the Avp/Oxt complex to chromosome 2 in the mouse. This map position confirms and extends themore » known region of linkage conservation between mouse chromosome 2 and human chromosome 20. 16 refs., 2 figs., 1 tab.« less

Tumor necrosis factor alpha converting enzyme: an encouraging target for various inflammatory disorders.

PubMed

Bahia, Malkeet S; Silakari, Om

2010-05-01

Tumor necrosis factor alpha is one of the most common pro-inflammatory cytokines responsible for various inflammatory disorders. It plays an important role in the origin and progression of rheumatoid arthritis and also in other autoimmune disease conditions. Some anti-tumor necrosis factor alpha antibodies like Enbrel, Humira and Remicade have been successfully used in these disease conditions as antagonists of tumor necrosis factor alpha. Inhibition of generation of active form of tumor necrosis factor alpha is a promising therapy for various inflammatory disorders. Therefore, the inhibition of an enzyme (tumor necrosis factor alpha converting enzyme), which is responsible for processing inactive form of tumor necrosis factor alpha into its active soluble form, is an encouraging target. Many tumor necrosis factor alpha converting enzyme inhibitors have been the candidates of clinical trials but none of them have reached in to the market because of their broad spectrum inhibitory activity for other matrix metalloproteases. Selectivity of tumor necrosis factor alpha converting enzyme inhibition over matrix metalloproteases is of utmost importance. If selectivity is achieved successfully, side-effects can be over-ruled and this approach may become a novel therapy for treatment of rheumatoid arthritis and other inflammatory disorders. This cytokine not only plays a pivotal role in inflammatory conditions but also in some cancerous conditions. Thus, successful targeting of tumor necrosis factor alpha converting enzyme may result in multifunctional therapy.

Type 1 diabetes in children is not a predisposing factor for oral yeast colonization.

PubMed

Costa, Ana L; Silva, Branca M A; Soares, Rui; Mota, Diana; Alves, Vera; Mirante, Alice; Ramos, João C; Maló de Abreu, João; Santos-Rosa, Manuel; Caramelo, Francisco; Gonçalves, Teresa

2017-06-01

Type 1 diabetes mellitus (T1D) is considered a risk factor associated with oral yeast infections. The aim of this study was to evaluate the yeast oral carriage (in saliva and mucosal surface) of children with T1D and potential relation with host factors, particularly the subset of CD4+ T cells. Yeasts were quantified and identified in stimulated saliva and in cheek mucosal swabs of 133 diabetic T1D and 72 healthy control subjects. Salivary lymphocytes were quantified using flow cytometry. The presence of yeasts in the oral cavity (60% of total patients) was not affected by diabetes, metabolic control, duration of the disease, salivary flow rate or saliva buffer capacity, by age, sex, place of residence, number of daily meals, consumption of sweets or frequency of tooth brushing. Candida albicans was the most prevalent yeast species, but a higher number of yeast species was isolated in nondiabetics. T1D children with HbA1c ≤ 7.5 (metabolically controlled) presented higher number of CD4+ T salivary subsets when compared with the other groups of children (non-diabetic and nonmetabolically controlled) and also presented the highest number of individuals without oral yeast colonization. In conclusion, T1D does not predisposes for increased oral yeast colonization and a higher number of salivary CD4+T cells seems to result in the absence of oral colonization by yeasts. © The Author 2016. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Biogenesis of lysosomal enzymes in the alpha-glucosidase II-deficient modA mutant of Dictyostelium discoideum: retention of alpha-1,3-linked glucose on N-linked oligosaccharides delays intracellular transport but does not alter sorting of alpha-mannosidase or beta-glucosidase.

PubMed

Ebert, D L; Bush, J M; Dimond, R L; Cardelli, J A

1989-09-01

The endoplasmic reticulum-localized enzyme alpha-glucosidase II is responsible for removing the two alpha-1,3-linked glucose residues from N-linked oligosaccharides of glycoproteins. This activity is missing in the modA mutant strain, M31, of Dictyostelium discoideum. Results from both radiolabeled pulse-chase and subcellular fractionation experiments indicate that this deficiency did not prevent intracellular transport and proteolytic processing of the lysosomal enzymes, alpha-mannosidase and beta-glucosidase. However, the rate at which the glucosylated precursors left the rough endoplasmic reticulum was several-fold slower than the rate at which the wild-type precursors left this compartment. Retention of glucose residues did not disrupt the binding of the precursor forms of the enzymes with intracellular membranes, indicating that the delay in movement of proteins from the ER did not result from lack of association with membranes. However, the mutant alpha-mannosidase precursor contained more trypsin-sensitive sites than did the wild-type precursor, suggesting that improper folding of precursor molecules might account for the slow rate of transport to the Golgi complex. Percoll density gradient fractionation of extracts prepared from M31 cells indicated that the proteolytically processed mature forms of alpha-mannosidase and beta-glucosidase were localized to lysosomes. Finally, the mutation in M31 may have other, more dramatic, effects on the lysosomal system since two enzymes, N-acetylglucosaminidase and acid phosphatase, were secreted much less efficiently from lysosomal compartments by the mutant strain.

Biogenesis of the Saccharomyces cerevisiae Pheromone a-Factor, from Yeast Mating to Human Disease

PubMed Central

Barrowman, Jemima

2012-01-01

Summary: The mating pheromone a-factor secreted by Saccharomyces cerevisiae is a farnesylated and carboxylmethylated peptide and is unusually hydrophobic compared to other extracellular signaling molecules. Mature a-factor is derived from a precursor with a C-terminal CAAX motif that directs a series of posttranslational reactions, including prenylation, endoproteolysis, and carboxylmethylation. Historically, a-factor has served as a valuable model for the discovery and functional analysis of CAAX-processing enzymes. In this review, we discuss the three modules comprising the a-factor biogenesis pathway: (i) the C-terminal CAAX-processing steps carried out by Ram1/Ram2, Ste24 or Rce1, and Ste14; (ii) two sequential N-terminal cleavage steps, mediated by Ste24 and Axl1; and (iii) export by a nonclassical mechanism, mediated by the ATP binding cassette (ABC) transporter Ste6. The small size and hydrophobicity of a-factor present both challenges and advantages for biochemical analysis, as discussed here. The enzymes involved in a-factor biogenesis are conserved from yeasts to mammals. Notably, studies of the zinc metalloprotease Ste24 in S. cerevisiae led to the discovery of its mammalian homolog ZMPSTE24, which cleaves the prenylated C-terminal tail of the nuclear scaffold protein lamin A. Mutations that alter ZMPSTE24 processing of lamin A in humans cause the premature-aging disease progeria and related progeroid disorders. Intriguingly, recent evidence suggests that the entire a-factor pathway, including all three biogenesis modules, may be used to produce a prenylated, secreted signaling molecule involved in germ cell migration in Drosophila. Thus, additional prenylated signaling molecules resembling a-factor, with as-yet-unknown roles in metazoan biology, may await discovery. PMID:22933563

C/EBP beta regulation of the tumor necrosis factor alpha gene.

PubMed Central

Pope, R M; Leutz, A; Ness, S A

1994-01-01

Activated macrophages contribute to chronic inflammation by the secretion of cytokines and proteinases. Tumor necrosis factor alpha (TNF alpha) is particularly important in this process because of its ability to regulate other inflammatory mediators in an autocrine and paracrine fashion. The mechanism(s) responsible for the cell type-specific regulation of TNF alpha is not known. We present data to show that the expression of TNF alpha is regulated by the transcription factor C/EBP beta (NF-IL6). C/EBP beta activated the TNF alpha gene promoter in cotransfection assays and bound to it at a site which failed to bind the closely related protein C/EBP alpha. Finally, a dominant-negative version of C/EBP beta blocked TNF alpha promoter activation in myeloid cells. Our results implicate C/EBP beta as an important regulator of TNF alpha by myelomonocytic cells. Images PMID:7929820

Oxygen Consumption by Postfermentation Wine Yeast Lees: Factors Affecting Its Rate and Extent under Oenological Conditions

PubMed Central

Müller, Jonas; Schmidt, Dominik

2016-01-01

Summary Postfermentation wine yeast lees show antioxidant properties based on their ability to consume dissolved oxygen. The oxygen consumption capacity of suspended yeast lees obtained after fermentations with six commercial active dry yeast strains was investigated in model, white and red wines using fluorescence-based oxygen sensors operating in a nondestructive way. In model solution, the oxygen consumption rate of yeast lees was shown to depend on their amount, yeast strain, sulfur dioxide and temperature. It is slightly lower in red than in white wines. It is strongly decreased by current levels of free sulfur dioxide, thus excluding the complementary use of both as antioxidants in wine. However, in 25 randomly sampled white wines produced under commercial conditions, the rate and extent of oxygen consumption during the first six months of postfermentation had no significant correlation with any of these interacting factors, making it difficult to predict the actual antioxidant effect of yeast lees. In these wines, yeast lees consumed 0 to 47% of the dissolved oxygen. Although total oxygen consumption capacity of yeast lees is not a limiting factor under commercial winemaking conditions, their oxygen consumption proceeds at a limited rate that reduces but cannot totally prevent concomitant chemical oxidation of the wine. PMID:28115896

Oxygen Consumption by Postfermentation Wine Yeast Lees: Factors Affecting Its Rate and Extent under Oenological Conditions.

PubMed

Schneider, Volker; Müller, Jonas; Schmidt, Dominik

2016-12-01

Postfermentation wine yeast lees show antioxidant properties based on their ability to consume dissolved oxygen. The oxygen consumption capacity of suspended yeast lees obtained after fermentations with six commercial active dry yeast strains was investigated in model, white and red wines using fluorescence-based oxygen sensors operating in a nondestructive way. In model solution, the oxygen consumption rate of yeast lees was shown to depend on their amount, yeast strain, sulfur dioxide and temperature. It is slightly lower in red than in white wines. It is strongly decreased by current levels of free sulfur dioxide, thus excluding the complementary use of both as antioxidants in wine. However, in 25 randomly sampled white wines produced under commercial conditions, the rate and extent of oxygen consumption during the first six months of postfermentation had no significant correlation with any of these interacting factors, making it difficult to predict the actual antioxidant effect of yeast lees. In these wines, yeast lees consumed 0 to 47% of the dissolved oxygen. Although total oxygen consumption capacity of yeast lees is not a limiting factor under commercial winemaking conditions, their oxygen consumption proceeds at a limited rate that reduces but cannot totally prevent concomitant chemical oxidation of the wine.

Deteriorated Stress Response in Stationary-Phase Yeast: Sir2 and Yap1 Are Essential for Hsf1 Activation by Heat Shock and Oxidative Stress, Respectively

PubMed Central

Cohen, Aviv; Bar-Nun, Shoshana

2014-01-01

Stationary-phase cultures have been used as an important model of aging, a complex process involving multiple pathways and signaling networks. However, the molecular processes underlying stress response of non-dividing cells are poorly understood, although deteriorated stress response is one of the hallmarks of aging. The budding yeast Saccharomyces cerevisiae is a valuable model organism to study the genetics of aging, because yeast ages within days and are amenable to genetic manipulations. As a unicellular organism, yeast has evolved robust systems to respond to environmental challenges. This response is orchestrated largely by the conserved transcription factor Hsf1, which in S. cerevisiae regulates expression of multiple genes in response to diverse stresses. Here we demonstrate that Hsf1 response to heat shock and oxidative stress deteriorates during yeast transition from exponential growth to stationary-phase, whereas Hsf1 activation by glucose starvation is maintained. Overexpressing Hsf1 does not significantly improve heat shock response, indicating that Hsf1 dwindling is not the major cause for Hsf1 attenuated response in stationary-phase yeast. Rather, factors that participate in Hsf1 activation appear to be compromised. We uncover two factors, Yap1 and Sir2, which discretely function in Hsf1 activation by oxidative stress and heat shock. In Δyap1 mutant, Hsf1 does not respond to oxidative stress, while in Δsir2 mutant, Hsf1 does not respond to heat shock. Moreover, excess Sir2 mimics the heat shock response. This role of the NAD+-dependent Sir2 is supported by our finding that supplementing NAD+ precursors improves Hsf1 heat shock response in stationary-phase yeast, especially when combined with expression of excess Sir2. Finally, the combination of excess Hsf1, excess Sir2 and NAD+ precursors rejuvenates the heat shock response. PMID:25356557

Deteriorated stress response in stationary-phase yeast: Sir2 and Yap1 are essential for Hsf1 activation by heat shock and oxidative stress, respectively.

PubMed

Nussbaum, Inbal; Weindling, Esther; Jubran, Ritta; Cohen, Aviv; Bar-Nun, Shoshana

2014-01-01

Stationary-phase cultures have been used as an important model of aging, a complex process involving multiple pathways and signaling networks. However, the molecular processes underlying stress response of non-dividing cells are poorly understood, although deteriorated stress response is one of the hallmarks of aging. The budding yeast Saccharomyces cerevisiae is a valuable model organism to study the genetics of aging, because yeast ages within days and are amenable to genetic manipulations. As a unicellular organism, yeast has evolved robust systems to respond to environmental challenges. This response is orchestrated largely by the conserved transcription factor Hsf1, which in S. cerevisiae regulates expression of multiple genes in response to diverse stresses. Here we demonstrate that Hsf1 response to heat shock and oxidative stress deteriorates during yeast transition from exponential growth to stationary-phase, whereas Hsf1 activation by glucose starvation is maintained. Overexpressing Hsf1 does not significantly improve heat shock response, indicating that Hsf1 dwindling is not the major cause for Hsf1 attenuated response in stationary-phase yeast. Rather, factors that participate in Hsf1 activation appear to be compromised. We uncover two factors, Yap1 and Sir2, which discretely function in Hsf1 activation by oxidative stress and heat shock. In Δyap1 mutant, Hsf1 does not respond to oxidative stress, while in Δsir2 mutant, Hsf1 does not respond to heat shock. Moreover, excess Sir2 mimics the heat shock response. This role of the NAD+-dependent Sir2 is supported by our finding that supplementing NAD+ precursors improves Hsf1 heat shock response in stationary-phase yeast, especially when combined with expression of excess Sir2. Finally, the combination of excess Hsf1, excess Sir2 and NAD+ precursors rejuvenates the heat shock response.

Rrp5p, Noc1p and Noc2p form a protein module which is part of early large ribosomal subunit precursors in S. cerevisiae

PubMed Central

Hierlmeier, Thomas; Merl, Juliane; Sauert, Martina; Perez-Fernandez, Jorge; Schultz, Patrick; Bruckmann, Astrid; Hamperl, Stephan; Ohmayer, Uli; Rachel, Reinhard; Jacob, Anja; Hergert, Kristin; Deutzmann, Rainer; Griesenbeck, Joachim; Hurt, Ed; Milkereit, Philipp; Baßler, Jochen; Tschochner, Herbert

2013-01-01

Eukaryotic ribosome biogenesis requires more than 150 auxiliary proteins, which transiently interact with pre-ribosomal particles. Previous studies suggest that several of these biogenesis factors function together as modules. Using a heterologous expression system, we show that the large ribosomal subunit (LSU) biogenesis factor Noc1p of Saccharomyces cerevisiae can simultaneously interact with the LSU biogenesis factor Noc2p and Rrp5p, a factor required for biogenesis of the large and the small ribosomal subunit. Proteome analysis of RNA polymerase-I-associated chromatin and chromatin immunopurification experiments indicated that all members of this protein module and a specific set of LSU biogenesis factors are co-transcriptionally recruited to nascent ribosomal RNA (rRNA) precursors in yeast cells. Further ex vivo analyses showed that all module members predominantly interact with early pre-LSU particles after the initial pre-rRNA processing events have occurred. In yeast strains depleted of Noc1p, Noc2p or Rrp5p, levels of the major LSU pre-rRNAs decreased and the respective other module members were associated with accumulating aberrant rRNA fragments. Therefore, we conclude that the module exhibits several binding interfaces with pre-ribosomes. Taken together, our results suggest a co- and post-transcriptional role of the yeast Rrp5p–Noc1p–Noc2p module in the structural organization of early LSU precursors protecting them from non-productive RNase activity. PMID:23209026

Evidence that N-acetylcysteine inhibits TNF-alpha-induced cerebrovascular endothelin-1 upregulation via inhibition of mitogen- and stress-activated protein kinase.

PubMed

Sury, Matthias D; Frese-Schaper, Manuela; Mühlemann, Miranda K; Schulthess, Fabienne T; Blasig, Ingolf E; Täuber, Martin G; Shaw, Sidney G; Christen, Stephan

2006-11-01

N-acetylcysteine (NAC) is neuroprotective in animal models of acute brain injury such as caused by bacterial meningitis. However, the mechanism(s) by which NAC exerts neuroprotection is unclear. Gene expression of endothelin-1 (ET-1), which contributes to cerebral blood flow decline in acute brain injury, is partially regulated by reactive oxygen species, and thus a potential target of NAC. We therefore examined the effect of NAC on tumor necrosis factor (TNF)-alpha-induced ET-1 production in cerebrovascular endothelial cells. NAC dose dependently inhibited TNF-alpha-induced preproET-1 mRNA upregulation and ET-1 protein secretion, while upregulation of inducible nitric oxide synthase (iNOS) was unaffected. Intriguingly, NAC had no effect on the initial activation (i.e., IkappaB degradation, nuclear p65 translocation, and Ser536 phosphorylation) of NF-kappaB by TNF-alpha. However, transient inhibition of NF-kappaB DNA binding suggested that NAC may inhibit ET-1 upregulation by inhibiting (a) parallel pathway(s) necessary for full transcriptional activation of NF-kappaB-mediated ET-1 gene expression. Similar to NAC, the MEK1/2 inhibitor U0126, the p38 inhibitor SB203580, and the protein kinase inhibitor H-89 selectively inhibited ET-1 upregulation without affecting nuclear p65 translocation, suggesting that NAC inhibits ET-1 upregulation via inhibition of mitogen- and stress-activated protein kinase (MSK). Supporting this notion, cotreatment with NAC inhibited the TNF-alpha-induced rise in MSK1 and MSK2 kinase activity, while siRNA knock-down experiments showed that MSK2 is the predominant isoform involved in TNF-alpha-induced ET-1 upregulation.

Global Analysis of Transcription Factor-Binding Sites in Yeast Using ChIP-Seq

PubMed Central

Lefrançois, Philippe; Gallagher, Jennifer E. G.; Snyder, Michael

2016-01-01

Transcription factors influence gene expression through their ability to bind DNA at specific regulatory elements. Specific DNA-protein interactions can be isolated through the chromatin immunoprecipitation (ChIP) procedure, in which DNA fragments bound by the protein of interest are recovered. ChIP is followed by high-throughput DNA sequencing (Seq) to determine the genomic provenance of ChIP DNA fragments and their relative abundance in the sample. This chapter describes a ChIP-Seq strategy adapted for budding yeast to enable the genome-wide characterization of binding sites of transcription factors (TFs) and other DNA-binding proteins in an efficient and cost-effective way. Yeast strains with epitope-tagged TFs are most commonly used for ChIP-Seq, along with their matching untagged control strains. The initial step of ChIP involves the cross-linking of DNA and proteins. Next, yeast cells are lysed and sonicated to shear chromatin into smaller fragments. An antibody against an epitope-tagged TF is used to pull down chromatin complexes containing DNA and the TF of interest. DNA is then purified and proteins degraded. Specific barcoded adapters for multiplex DNA sequencing are ligated to ChIP DNA. Short DNA sequence reads (28–36 base pairs) are parsed according to the barcode and aligned against the yeast reference genome, thus generating a nucleotide-resolution map of transcription factor-binding sites and their occupancy. PMID:25213249

A ribosome-dependent GTPase from yeast distinct from elongation factor 2.

PubMed Central

Skogerson, L; Wakatama, E

1976-01-01

Three proteins required for poly(U)-directed polyphenylalanine synthesis have been separated from yeast. Two of the factors correspond to the elongation factors 1 and 2 described for other eukaryotic systems, according to the criteria of phenylalanyl-tRNA binding and diphtheria toxin-catalyzed ADP-ribosylation. The third protein, while absolutely required for polyphenylalanine synthesis, was a more active ribosome-dependent GTPase than elongation factor 2. PMID:174100

Prediction of alpha factor values for fine pore aeration systems.

PubMed

Gillot, S; Héduit, A

2008-01-01

The objective of this work was to analyse the impact of different geometric and operating parameters on the alpha factor value for fine bubble aeration systems equipped with EPDM membrane diffusers. Measurements have been performed on nitrifying plants operating under extended aeration and treating mainly domestic wastewater. Measurements performed on 14 nitrifying plants showed that, for domestic wastewater treatment under very low F/M ratios, the alpha factor is comprised between 0.44 and 0.98. A new composite variable (the Equivalent Contact Time, ECT) has been defined and makes it possible for a given aeration tank, knowing the MCRT, the clean water oxygen transfer coefficient and the supplied air flow rate, to predict the alpha factor value. ECT combines the effect on mass transfer of all generally accepted factors affecting oxygen transfer performances (air flow rate, diffuser submergence, horizontal flow). (c) IWA Publishing 2008.

Engineering tolerance to industrially relevant stress factors in yeast cell factories.

PubMed

Deparis, Quinten; Claes, Arne; Foulquié-Moreno, Maria R; Thevelein, Johan M

2017-06-01

The main focus in development of yeast cell factories has generally been on establishing optimal activity of heterologous pathways and further metabolic engineering of the host strain to maximize product yield and titer. Adequate stress tolerance of the host strain has turned out to be another major challenge for obtaining economically viable performance in industrial production. Although general robustness is a universal requirement for industrial microorganisms, production of novel compounds using artificial metabolic pathways presents additional challenges. Many of the bio-based compounds desirable for production by cell factories are highly toxic to the host cells in the titers required for economic viability. Artificial metabolic pathways also turn out to be much more sensitive to stress factors than endogenous pathways, likely because regulation of the latter has been optimized in evolution in myriads of environmental conditions. We discuss different environmental and metabolic stress factors with high relevance for industrial utilization of yeast cell factories and the experimental approaches used to engineer higher stress tolerance. Improving stress tolerance in a predictable manner in yeast cell factories should facilitate their widespread utilization in the bio-based economy and extend the range of products successfully produced in large scale in a sustainable and economically profitable way. © FEMS 2017.

Engineering tolerance to industrially relevant stress factors in yeast cell factories

PubMed Central

Deparis, Quinten; Claes, Arne; Foulquié-Moreno, Maria R.

2017-01-01

Abstract The main focus in development of yeast cell factories has generally been on establishing optimal activity of heterologous pathways and further metabolic engineering of the host strain to maximize product yield and titer. Adequate stress tolerance of the host strain has turned out to be another major challenge for obtaining economically viable performance in industrial production. Although general robustness is a universal requirement for industrial microorganisms, production of novel compounds using artificial metabolic pathways presents additional challenges. Many of the bio-based compounds desirable for production by cell factories are highly toxic to the host cells in the titers required for economic viability. Artificial metabolic pathways also turn out to be much more sensitive to stress factors than endogenous pathways, likely because regulation of the latter has been optimized in evolution in myriads of environmental conditions. We discuss different environmental and metabolic stress factors with high relevance for industrial utilization of yeast cell factories and the experimental approaches used to engineer higher stress tolerance. Improving stress tolerance in a predictable manner in yeast cell factories should facilitate their widespread utilization in the bio-based economy and extend the range of products successfully produced in large scale in a sustainable and economically profitable way. PMID:28586408

Combined metabolic engineering of precursor and co-factor supply to increase α-santalene production by Saccharomyces cerevisiae

PubMed Central

2012-01-01

Background Sesquiterpenes are a class of natural products with a diverse range of attractive industrial proprieties. Due to economic difficulties of sesquiterpene production via extraction from plants or chemical synthesis there is interest in developing alternative and cost efficient bioprocesses. The hydrocarbon α-santalene is a precursor of sesquiterpenes with relevant commercial applications. Here, we construct an efficient Saccharomyces cerevisiae cell factory for α-santalene production. Results A multistep metabolic engineering strategy targeted to increase precursor and cofactor supply was employed to manipulate the yeast metabolic network in order to redirect carbon toward the desired product. To do so, genetic modifications were introduced acting to optimize the farnesyl diphosphate branch point, modulate the mevalonate pathway, modify the ammonium assimilation pathway and enhance the activity of a transcriptional activator. The approach employed resulted in an overall α-santalene yield of a 0.0052 Cmmol (Cmmol glucose)-1 corresponding to a 4-fold improvement over the reference strain. This strategy, combined with a specifically developed continuous fermentation process, led to a final α-santalene productivity of 0.036 Cmmol (g biomass)-1 h-1. Conclusions The results reported in this work illustrate how the combination of a metabolic engineering strategy with fermentation technology optimization can be used to obtain significant amounts of the high-value sesquiterpene α-santalene. This represents a starting point toward the construction of a yeast “sesquiterpene factory” and for the development of an economically viable bio-based process that has the potential to replace the current production methods. PMID:22938570

The response to inositol: regulation of glycerolipid metabolism and stress response signaling in yeast

PubMed Central

Henry, Susan A.; Gaspar, Maria L.; Jesch, Stephen A.

2014-01-01

This article focuses on discoveries of the mechanisms governing the regulation of glycerolipid metabolism and stress response signaling in response to the phospholipid precursor, inositol. The regulation of glycerolipid lipid metabolism in yeast in response to inositol is highly complex, but increasingly well understood, and the roles of individual lipids in stress response are also increasingly well characterized. Discoveries that have emerged over several decades of genetic, molecular and biochemical analyses of metabolic, regulatory and signaling responses of yeast cells, both mutant and wild type, to the availability of the phospholipid precursor, inositol are discussed. PMID:24418527

Biosynthesis of ependymins from goldfish brain.

PubMed

Königstorfer, A; Sterrer, S; Hoffmann, W

1989-08-15

Ependymins beta and gamma constitute a novel family of secretory proteins in the extracellular fluid of goldfish brain. Here we demonstrate that at least two different transcripts exist in goldfish brain differing mainly in the length of their 3' noncoding regions but encoding very similar precursors for ependymins. Both precursors consist of 216 amino acid residues including two potential N-glycosylation sites. Prepro-ependymin-I is the main but not the only precursor of ependymin beta, whereas prepro-ependymin-II is preferentially processed to ependymin gamma. This is in line with our results showing that both ependymins beta and gamma represent different glycoforms with very similar protein backbones. Additionally, we show that both ependymins share the same C-terminal ends indicating that ependymin gamma is not a proteolysis product of ependymin beta. We also demonstrate that processing at three internal pairs of basic residues does not occur in either ependymin.

Molecular cloning and expression of a gene for a factor which stabilizes formation of inhibitor-mitochondrial ATPase complex from Saccharomyces cerevisiae.

PubMed

Akashi, A; Yoshida, Y; Nakagoshi, H; Kuroki, K; Hashimoto, T; Tagawa, K; Imamoto, F

1988-10-01

Stabilizing factor, a 9 kDa protein, stabilizes and facilitates formation of the complex between mitochondrial ATP synthase and its intrinsic inhibitor protein. A clone containing the gene encoding the 9 kDa protein was selected from a yeast genomic library to determine the structure of its precursor protein. As deduced from the nucleotide sequence, the precursor of the yeast 9 kDa stabilizing factor contains 86 amino acid residues and has a molecular weight of 10,062. From the predicted sequence we infer that the stabilizing factor precursor contains a presequence of 23 amino acid residues at its amino terminus. We also used S1 mapping to determine the initiation site of transcription under glucose-repressed or derepressed conditions. These experiments suggest that transcription of this gene starts at three different sites and that only one of them is not affected by the presence of glucose.

Serum and Urinary Levels of Tumor Necrosis Factor-Alpha in Renal Transplant Patients.

PubMed

Senturk Ciftci, Hayriye; Demir, Erol; Savran Karadeniz, Meltem; Tefik, Tzevat; Yazici, Halil; Nane, Ismet; Savran Oguz, Fatma; Aydin, Filiz; Turkmen, Aydin

2017-12-18

Allograft rejection is an important cause of early and long-term graft loss in kidney transplant recipients. Tumor necrosis factor-alpha promotes T-cell activation, the key reaction leading to allograft rejection. Here, we investigated whether serum and urinary tumor necrosis factor-alpha levels can predict allograft rejection. This study included 65 living related-donor renal transplant recipients with mean follow-up of 26 ± 9 months. Serum and urinary tumor necrosis factor-alpha levels were measured at pretransplant and at posttransplant time points (days 1 and 7 and months 3 and 6); serum creatinine levels were also monitored during posttransplant follow-up. Standard enzyme-linked immunoabsorbent assay was used to detect tumor necrosis factor-alpha levels. Clinical variables were monitored. Nine of 65 patients (13.8%) had biopsy-proven rejection during follow-up. Preoperative serum and urinary tumor necrosis factor-alpha levels were not significantly different when we compared patients with and without rejection. Serum tumor necrosis factor-alpha levels (in pg/mL) were significantly higher in the allograft rejection versus nonrejection group at day 7 (11.5 ± 4.7 vs 15.4 ± 5.8; P = .029) and month 1 (11.1 ± 4.8 vs 17.8 ± 10.9; P =.003). Urinary tumor necrosis factor-alpha levels (in pg/mL) were also elevated in the allograft rejection versus the nonrejection group at days 1 (10.2 ± 2.5 vs 14.1 ± 6.8; P = .002) and 7 (9.8 ± 2.2 vs 14.5 ± 2.7; P < .001) and at months 1 (8.0 ± 1.7 vs 11.8 ± 2.4; P < .001), 3 (7.7 ± 1.6 vs 9.6 ± 1.7; P = .002), and 6 (7.4 ± 1.6 vs 8.9 ± 0.9; P = .005). Our preliminary findings suggest that tumor necrosis factor-alpha has a role in diagnosing renal transplant rejection. Serum and urinary tumor necrosis factor-alpha levels may be a possible predictor for allograft rejection.

Cardiomyocyte-released factors stimulate oligodendrocyte precursor cells proliferation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kuroda, Mariko; Muramatsu, Rieko; Precursory Research for Embryonic Science and Technology

The heart produces multiple diffusible factors that are involved in a number of physiological processes, but the action of these factors on the central nervous system is not well understood. In this study, we found that one or more factors released by cardiomyocytes promote oligodendrocyte precursor cell (OPC) proliferation in vitro. Mouse OPCs co-cultured with mouse cardiomyocytes showed higher proliferative ability than OPCs cultured alone. In addition, cardiomyocyte-conditioned media was sufficient to promote OPC proliferation. The phosphorylation of phosphatidylinositol (PI) 3-kinase and extracellular signal-regulated kinase (ERK) in OPCs is necessary for the enhancement of OPC proliferation by cardiomyocyte-conditioned media. These datamore » indicate that heart-derived factors have the ability to directly regulate the function of central nervous system (CNS) cells.« less

Kruppel-like factor 2 inhibits hypoxia-inducible factor 1alpha expression and function in the endothelium.

PubMed

Kawanami, Daiji; Mahabeleshwar, Ganapati H; Lin, Zhiyong; Atkins, G Brandon; Hamik, Anne; Haldar, Saptarsi M; Maemura, Koji; Lamanna, Joseph C; Jain, Mukesh K

2009-07-31

Hypoxia-inducible factor 1 (HIF-1) is a central regulator of the hypoxic response in many cell types. In endothelial cells, HIF-1 induces the expression of key proangiogenic factors to promote angiogenesis. Recent studies have identified Kruppel-like factor 2 (KLF2) as a potent inhibitor of angiogenesis. However, the role of KLF2 in regulating HIF-1 expression and function has not been evaluated. KLF2 expression was induced acutely by hypoxia in endothelial cells. Adenoviral overexpression of KLF2 inhibited hypoxia-induced expression of HIF-1alpha and its target genes such as interleukin 8, angiopoietin-2, and vascular endothelial growth factor in endothelial cells. Conversely, knockdown of KLF2 increased expression of HIF-1alpha and its targets. Furthermore, KLF2 inhibited hypoxia-induced endothelial tube formation, whereas endothelial cells from mice with haploinsufficiency of KLF2 showed increased tube formation in response to hypoxia. Consistent with this ex vivo observation, KLF2 heterozygous mice showed increased microvessel density in the brain. Mechanistically, KLF2 promoted HIF-1alpha degradation in a von Hippel-Lindau protein-independent but proteasome-dependent manner. Finally, KLF2 disrupted the interaction between HIF-1alpha and its chaperone Hsp90, suggesting that KLF2 promotes degradation of HIF-1alpha by affecting its folding and maturation. These observations identify KLF2 as a novel inhibitor of HIF-1alpha expression and function. Therefore, KLF2 may be a target for modulating the angiogenic response in disease states.

Tumor necrosis factor-alpha converting enzyme in the human placenta throughout gestation.

PubMed

Hung, Tai-Ho; Chen, Szu-Fu; Hsieh, Ching-Chang; Hsu, Jenn-Jeih; Li, Meng-Jen; Yeh, Yi-Lin; Hsieh, T'sang-T'ang

2008-02-01

Ectodomain shedding of epidermal growth factor receptor ligands such as transforming growth factor- alpha (TGF-alpha), heparin-binding epidermal growth factor-like growth factor (HBEGF), and amphiregulin (AREG) is considered to be important during implantation. Tumor necrosis factor-alpha converting enzyme (TACE) has been suggested as the major sheddase for these molecules. The objectives of this study are (1) to characterize the expression of TACE in the human placenta throughout gestation; (2) to determine the association between the expression of TACE with TGF-alpha, HBEGF, and AREG; (3) to ascertain whether TACE mediates TGF-alpha, HBEGF, and AREG shedding; and (4) to examine the effect of hypoxia on the expression of TACE. By analyzing a total of 55 villous samples representing different gestational ages, the authors found that TACE was continuously expressed in the placentas throughout gestation and that the levels of TACE were positively correlated with the levels of TGF-alpha, HBEGF, and AREG. Preadministration of a TACE inhibitor in villous explant cultures or transfection of cytotrophoblastic cells with TACE-specific small interference RNA decreased the shedding of HBEGF and AREG. Moreover, hypoxia (2% O(2)) caused an increase in the levels of TACE mRNA and protein in villous explants and primary cytotrophoblastic cells in vitro. These results indicate that oxygen regulates the expression of TACE and that TACE may be important for placental development during human pregnancy.

Immunoreactive transforming growth factor alpha is commonly present in colorectal neoplasia.

PubMed Central

Tanaka, S.; Imanishi, K.; Yoshihara, M.; Haruma, K.; Sumii, K.; Kajiyama, G.; Akamatsu, S.

1991-01-01

Surgical specimens from 19 patients with invasive colorectal cancers and 12 specimens of normal mucosa from the same patients were examined immunohistochemically for the production of the immunoreactive (IR-) transforming growth factor (TGF)-alpha and IR-epidermal growth factor (EGF) with an anti-TGF-alpha monoclonal antibody (MAb) OAL-MTG01 and anti-EGF MAb KEM-10. Immunoreactive TGF-alpha was detected in 16 (84.2%) of 19 colorectal cancers. In contrast, there was no IR-TGF-alpha in the gland cells of normal mucosa. Immunoreactive EGF was detected in 7 (36.8%) of 19 colorectal cancers and 1 (8.3%) of 12 cases of normal mucosa. The production of both IR-TGF-alpha and IR-EGF in colorectal cancer did not differ by histologic type and Dukes' stage. Immunoreactive TGF-alpha was detected at significantly higher incidence than IR-EGF in colorectal cancer. These results indicate that IR-TGF-alpha should prove valuable as a possible tumor marker in colorectal cancers, and it may be very useful in understanding the biology of colorectal cancer. Images Figure 2 Figure 3 Figure 4 Figure 5 PMID:1853928

A kinetic comparison of the processing and secretion of the alpha beta dimer and the uncombined alpha and beta subunits of chorionic gonadotropin synthesized by human choriocarcinoma cells.

PubMed

Peters, B P; Krzesicki, R F; Hartle, R J; Perini, F; Ruddon, R W

1984-12-25

Human choriocarcinoma cells (JAR) synthesize the alpha and beta subunits of the glycoprotein hormone chorionic gonadotropin (hCG) (R.W. Ruddon, C.A. Hanson, A. H. Bryan, G.J. Putterman, E.L. White, F. Perini, K. S. Meade, and P.H. Aldenderfer (1980) J. Biol. Chem. 255, 1000-1007). In addition to the hCG dimer (alpha beta), JAR cells secrete uncombined alpha and beta subunits into the culture medium (L.A. Cole, R.J. Hartle, J.A. Laferla, and R.W. Ruddon (1983) Endocrinology 113, 1176-1178). Pulse-chase studies with [35S]methionine or [3H]mannose were carried out in order to compare free alpha, free beta, and the alpha beta dimer with regard to the kinetics of synthesis, N-linked oligosaccharide processing, and secretion and to determine the kinetics of alpha-beta subunit combination. A panel of three antisera was used to immunoprecipitate directly the free subunits and the alpha beta dimer sequentially from the same cell lysates and culture media. The alpha subunit of hCG was synthesized in a slight molar excess (1.2-1.5-fold) over the beta subunit, and alpha beta dimer was rapidly formed by combination of the intracellular alpha and beta precursors. Dimer formation was already apparent in JAR cells following a 10-min biosynthetic labeling incubation with [35S]methionine. The combination of subunits ceased by 30 min of chase even though 51% of alpha and 44% of beta remained free within the cells. Combination of the alpha and beta precursors had occurred before their N-linked oligosaccharides were processed beyond the Man8GlcNAc2 structure. The initial trimming of glucosyl and mannosyl units from the high-mannose oligosaccharides of the hCG precursors occurred more rapidly for free alpha and CG-alpha than for free beta and CG-beta. JAR cells accumulated alpha precursors bearing mostly Man8GlcNAc2 units and beta precursors bearing Man8GlcNAc2 units that represent the substrates of the rate-limiting step in the secretory pathway. In spite of the fact that their N

Reconstitution of the yeast RNA polymerase III transcription system with all recombinant factors.

PubMed

Ducrot, Cécile; Lefebvre, Olivier; Landrieux, Emilie; Guirouilh-Barbat, Josée; Sentenac, André; Acker, Joel

2006-04-28

Transcription factor TFIIIC is a multisubunit complex required for promoter recognition and transcriptional activation of class III genes. We describe here the reconstitution of complete recombinant yeast TFIIIC and the molecular characterization of its two DNA-binding domains, tauA and tauB, using the baculovirus expression system. The B block-binding module, rtauB, was reconstituted with rtau138, rtau91, and rtau60 subunits. rtau131, rtau95, and rtau55 formed also a stable complex, rtauA, that displayed nonspecific DNA binding activity. Recombinant rTFIIIC was functionally equivalent to purified yeast TFIIIC, suggesting that the six recombinant subunits are necessary and sufficient to reconstitute a transcriptionally active TFIIIC complex. The formation and the properties of rTFIIIC-DNA complexes were affected by dephosphorylation treatments. The combination of complete recombinant rTFIIIC and rTFIIIB directed a low level of basal transcription, much weaker than with the crude B'' fraction, suggesting the existence of auxiliary factors that could modulate the yeast RNA polymerase III transcription system.

Breakup effects on alpha spectroscopic factors of 16O

NASA Astrophysics Data System (ADS)

Adhikari, S.; Basu, C.; Sugathan, P.; Jhinghan, A.; Behera, B. R.; Saneesh, N.; Kaur, G.; Thakur, M.; Mahajan, R.; Dubey, R.; Mitra, A. K.

2017-01-01

The triton angular distribution for the 12C(7Li,t)16O* reaction is measured at 20 MeV, populating discrete states of 16O. Continuum discretized coupled reaction channel calculations are used to to extract the alpha spectroscopic properties of 16O states instead of the distorted wave born approximation theory to include the effects of breakup on the transfer process. The alpha reduced width, spectroscopic factors and the asymptotic normalization constant (ANC) of 16O states are extracted. The error in the spectroscopic factor is about 35% and in that of the ANC about 27%.

Posttranslational processing of the prohormone-cleaving Kex2 protease in the Saccharomyces cerevisiae secretory pathway.

PubMed

Wilcox, C A; Fuller, R S

1991-10-01

The Kex2 protease of the yeast Saccharomyces cerevisiae is a prototypical eukaryotic prohormone-processing enzyme that cleaves precursors of secreted peptides at pairs of basic residues. Here we have established the pathway of posttranslational modification of Kex2 protein using immunoprecipitation of the biosynthetically pulse-labeled protein from a variety of wild-type and mutant yeast strains as the principal methodology. Kex2 protein is initially synthesized as a prepro-enzyme that undergoes cotranslational signal peptide cleavage and addition of Asn-linked core oligosaccharide and Ser/Thr-linked mannose in the ER. The earliest detectable species, I1 (approximately 129 kD), undergoes rapid amino-terminal proteolytic removal of a approximately 9-kD pro-segment yielding species I2 (approximately 120 kD) before arrival at the Golgi complex. Transport to the Golgi complex is marked by extensive elaboration of Ser/Thr-linked chains and minor modification of Asn-linked oligosaccharide. During the latter phase of its lifetime, Kex2 protein undergoes a gradual increase in apparent molecular weight. This final modification serves as a marker for association of Kex2 protease with a late compartment of the yeast Golgi complex in which it is concentrated about 27-fold relative to other secretory proteins.

Macrophage-induced angiogenesis is mediated by tumour necrosis factor-alpha.

PubMed

Leibovich, S J; Polverini, P J; Shepard, H M; Wiseman, D M; Shively, V; Nuseir, N

Macrophages are important in the induction of new blood vessel growth during wound repair, inflammation and tumour growth. We show here that tumour necrosis factor-alpha (TNF-alpha), a secretory product of activated macrophages that is believed to mediate tumour cytotoxicity, is a potent inducer of new blood vessel growth (angiogenesis). In vivo, TNF-alpha induces capillary blood vessel formation in the rat cornea and the developing chick chorioallantoic membrane at very low doses. In vitro, TNF-alpha stimulates chemotaxis of bovine adrenal capillary endothelial cells and induces cultures of these cells grown on type-1 collagen gels to form capillary-tube-like structures. The angiogenic activity produced by activated murine peritoneal macrophages is completely neutralized by a polyclonal antibody to TNF-alpha, suggesting immunological features are common to TNF-alpha and the protein responsible for macrophage-derived angiogenic activity. In inflammation and wound repair, TNF-alpha could augment repair by stimulating new blood vessel growth; in tumours, TNF-alpha might both stimulate tumour development by promoting vessel growth and participate in tumour destruction by direct cytotoxicity.

Transport of pyruvate and lactate in yeast mitochondria.

PubMed

Briquet, M

1977-02-07

Evidence for the existence of mediated transport of pyruvate and lactate in isolated mitochondria of Saccharomyces cerevisiae is presented. 1. The mitochondrial oxidation of pyruvate is specifically inhibited by the monocarboxylic oxoacids alpha-ketoisocaproate and by alpha-cyano-3-hydroxycinnamate, while pyruvate and malate dehydrogenases activities are not inhibited. 2. The stimulation of the mitochondrial oxidations of succinate, alpha-ketoglutarate and citrate by pyruvate are also inhibited by alpha-cyano-3-hydroxycinnamate. 3. The [14C]pyruvate uptake by yeast mitochondria follows saturation kinetics and is completely inhibited by alpha-cyano-3-hydroxycinnamate. 4. Large amplitude passive swellings of mitochondria of the wild type and of cytoplasmic rho- and rho-n mutants are induced by isoosmotic ammonium pyruvate and lactate. These pH-dependent swellings are inhibited by alpha-cyano-3-hydroxycinnamate suggesting that the carrier system is not coded by mitochondrial DNA.

Metabolism of the Fusarium mycotoxins zearalenone and deoxynivalenol by yeast strains of technological relevance.

PubMed

Böswald, C; Engelhardt, G; Vogel, H; Wallnöfer, P R

1995-01-01

The Fusarium mycotoxin zearalenone (ZEA), added at a level of 2 micrograms/ml, was reduced stereoselectively by cultures of Candida tropicalis, Torulaspora delbrückii, Zygosaccharomyces rouxii, and 7 Saccharomyces strains to both alpha- and beta-zearalenol. In contrast, only alpha-zearalenol was produced from ZEA by Pichia fermentans and several yeast strains of the genera Candida, Hansenula, Brettanomyces, Schizosaccharomyces, and Saccharomycopsis. No glucose conjugates of ZEA (zearalenone-4-beta-D-glucopyranoside) were detected. The trichothecene mycotoxin deoxynivalenol (DON) was not metabolized by any of the yeast strains that were used for analysis.

Expression of the Major Surface Antigen of Plasmodium knowlesi Sporozoites in Yeast

NASA Astrophysics Data System (ADS)

Sharma, Shobhona; Godson, G. Nigel

1985-05-01

The circumsporozoite protein, a surface antigen of the sporozoite stage of the monkey malarial parasite Plasmodium knowlesi, was expressed in the yeast Saccharomyces cerevisiae by using an expression vector containing the 5' regulatory region of the yeast alcohol dehydrogenase I gene. It was necessary to eliminate the entire 5' upstream region of the parasite DNA to obtain the expression of this protein. Only the circumsporozoite precursor protein was produced by the yeast transformants, as detected by immunoblotting. About 55 and 20 percent of the circumsporozoite protein produced in yeast was associated with the 25,000g and 150,000g particulate fractions, respectively. The protein could be solubilized in Triton X-100 and was stable in solubilized extracts.

Transforming growth factor-{alpha} enhances corneal epithelial cell migration by promoting EGFR recycling.

PubMed

McClintock, Jennifer L; Ceresa, Brian P

2010-07-01

PURPOSE. The goal of this study was to determine the molecular mechanism by which transforming growth factor-alpha (TGF-alpha) is a more potent activator of epidermal growth factor receptor (EGFR)-mediated corneal wound healing than epidermal growth factor (EGF). METHODS. Telomerase immortalized human corneal epithelial (hTCEpi) cells and primary human corneal epithelial cells were tested for their ability to migrate in response to EGF and TGF-alpha. In parallel, the endocytic trafficking of the EGFR in response to these same ligands was examined using indirect immunofluorescence, immunoblots, and radioligand binding. RESULTS. TGF-alpha, compared with EGF, is a more potent activator of corneal epithelial cell migration. Although both TGF-alpha and EGF were able to induce EGFR internalization and phosphorylation, only those receptors that were stimulated with EGF progressed to lysosomal degradation. EGFRs stimulated with TGF-alpha recycled back to the plasma membrane, where they could be reactivated with ligand. CONCLUSIONS. This study reveals that EGFR-mediated cell migration is limited by ligand-stimulated downregulation of the EGFR. This limitation can be overcome by treating cells with TGF-alpha because TGF-alpha stimulates EGFR endocytosis, but not degradation. After internalization of the TGF-alpha/EGFR complex, EGFR recycles back to the plasma membrane, where it can be restimulated. This sequence of events provides the receptor multiple opportunities for stimulation. Thus, stimulation with TGF-alpha prolongs EGFR signaling compared with EGF.

Interaction of cord factor (alpha, alpha'-trehalose-6,6'-dimycolate) with phospholipids.

PubMed

Crowe, L M; Spargo, B J; Ioneda, T; Beaman, B L; Crowe, J H

1994-08-24

We previously reported that cord factor (alpha,alpha'-trehalose-6,6'-dimycolate) isolated from Nocardia asteroides strain GUH-2 strongly inhibits fusion between unilamellar vesicles containing acidic phospholipid. We chose to study the effects of this molecule on liposome fusion since the presence of N. asteroides GUH-2 in the phagosomes of mouse macrophages had been shown to prevent phagosomal acidification and inhibit phagosome-lysosome fusion. A virtually non-virulent strain, N. asteroides 10905, does not prevent acidification or phagosome-lysosome fusion and, further, contains only trace amounts of cord factor. In the present paper, we have investigated the effects of cord factor on phospholipid bilayers that could be responsible for the inhibition of fusion. We show that cord factor increases molecular area, measured by isothermal compression of a monolayer film, in a mixed monolayer more than would be expected based in its individual contribution to molecular area. Cord factor, as well as other glycolipids investigated, increased the overall hydration of bilayers of dipalmitoylphosphatidylcholine by 50%, as estimated from the unfrozen water fraction measured by differential scanning calorimetry. The effect of calcium on this increased molecular area and headgroup hydration was measured by fluorescence anisotropy and FTIR spectroscopy of phosphatidylserine liposomes. Both techniques showed that cord factor, incorporated at 10 mol%, increased acyl chain disorder over controls in the presence of Ca2+. However, FTIR showed that cord factor did not prevent headgroup dehydration by the Ca2+. The other glycolipids tested did not prevent either the Ca(2+)-induced chain crystallization or headgroup dehydration of phosphatidylserine bilayers. These data point to a possible role of the bulky mycolic acids of cord factor in preventing Ca(2+)-induced fusion of liposomes containing acidic phospholipids.

Transforming growth factor-alpha (TGF-alpha) in a semisolid dosage form: preservative and vehicle selection.

PubMed

Tan, E L; Shah, H S; Leister, K J; Kozick, L M; Pasciak, P; Vanderlaan, R K; Yu, C D; Patel, B

1993-08-01

The selection of an ideal semisolid vehicle for growth factors presents a challenge. Some antimicrobial agents are known to delay wound healing. The objective of this investigation was to identify appropriate preservatives and vehicles for TGF-alpha. Criteria for acceptance are noninterference with the mitogenic activity of TGF-alpha as well as adequate product preservation. Vehicles considered were o/w creams, ointments, and a gel. Combinations of six preservatives were tested. Selection was determined using both microbial preservative challenge and TGF-alpha mitogenic assay. In the former, 10 species of microorganisms were inoculated into formulation samples. At selected time intervals, it was determined whether colonies decreased, increased, or remained constant. In the mitogenic assay, samples of either preservatives or formulation prototypes were introduced to TGF-alpha-stimulated fibroblast cell cultures. Mitogenesis was determined by measuring 3H-dThd uptake into newly synthesized DNA. As preservatives, sorbic acid and quaternium-15 appear to satisfy both selection criteria. A thermosetting gel appears most promising as vehicle.

Defective insulin secretion in hepatocyte nuclear factor 1alpha-deficient mice.

PubMed Central

Pontoglio, M; Sreenan, S; Roe, M; Pugh, W; Ostrega, D; Doyen, A; Pick, A J; Baldwin, A; Velho, G; Froguel, P; Levisetti, M; Bonner-Weir, S; Bell, G I; Yaniv, M; Polonsky, K S

1998-01-01

Mutations in the gene for the transcription factor hepatocyte nuclear factor (HNF) 1alpha cause maturity-onset diabetes of the young (MODY) 3, a form of diabetes that results from defects in insulin secretion. Since the nature of these defects has not been defined, we compared insulin secretory function in heterozygous [HNF-1alpha (+/-)] or homozygous [HNF-1alpha (-/-)] mice with null mutations in the HNF-1alpha gene with their wild-type littermates [HNF-1alpha (+/+)]. Blood glucose concentrations were similar in HNF-1alpha (+/+) and (+/-) mice (7.8+/-0.2 and 7.9+/-0.3 mM), but were significantly higher in the HNF-1alpha (-/-) mice (13.1+/-0.7 mM, P < 0.001). Insulin secretory responses to glucose and arginine in the perfused pancreas and perifused islets from HNF-1alpha (-/-) mice were < 15% of the values in the other two groups and were associated with similar reductions in intracellular Ca2+ responses. These defects were not due to a decrease in glucokinase or insulin gene transcription. beta cell mass adjusted for body weight was not reduced in the (-/-) animals, although pancreatic insulin content adjusted for pancreas weight was slightly lower (0.06+/-0.01 vs. 0.10+/-0.01 microg/mg, P < 0.01) than in the (+/+) animals. In summary, a null mutation in the HNF-1alpha gene in homozygous mice leads to diabetes due to alterations in the pathways that regulate beta cell responses to secretagogues including glucose and arginine. These results provide further evidence in support of a key role for HNF-1alpha in the maintenance of normal beta cell function. PMID:9593777

Metabolic analyses elucidate non-trivial gene targets for amplifying dihydroartemisinic acid production in yeast

PubMed Central

Misra, Ashish; Conway, Matthew F.; Johnnie, Joseph; Qureshi, Tabish M.; Lige, Bao; Derrick, Anne M.; Agbo, Eddy C.; Sriram, Ganesh

2013-01-01

Synthetic biology enables metabolic engineering of industrial microbes to synthesize value-added molecules. In this, a major challenge is the efficient redirection of carbon to the desired metabolic pathways. Pinpointing strategies toward this goal requires an in-depth investigation of the metabolic landscape of the organism, particularly primary metabolism, to identify precursor and cofactor availability for the target compound. The potent antimalarial therapeutic artemisinin and its precursors are promising candidate molecules for production in microbial hosts. Recent advances have demonstrated the production of artemisinin precursors in engineered yeast strains as an alternative to extraction from plants. We report the application of in silico and in vivo metabolic pathway analyses to identify metabolic engineering targets to improve the yield of the direct artemisinin precursor dihydroartemisinic acid (DHA) in yeast. First, in silico extreme pathway (ExPa) analysis identified NADPH-malic enzyme and the oxidative pentose phosphate pathway (PPP) as mechanisms to meet NADPH demand for DHA synthesis. Next, we compared key DHA-synthesizing ExPas to the metabolic flux distributions obtained from in vivo 13C metabolic flux analysis of a DHA-synthesizing strain. This comparison revealed that knocking out ethanol synthesis and overexpressing glucose-6-phosphate dehydrogenase in the oxidative PPP (gene YNL241C) or the NADPH-malic enzyme ME2 (YKL029C) are vital steps toward overproducing DHA. Finally, we employed in silico flux balance analysis and minimization of metabolic adjustment on a yeast genome-scale model to identify gene knockouts for improving DHA yields. The best strategy involved knockout of an oxaloacetate transporter (YKL120W) and an aspartate aminotransferase (YKL106W), and was predicted to improve DHA yields by 70-fold. Collectively, our work elucidates multiple non-trivial metabolic engineering strategies for improving DHA yield in yeast. PMID:23898325

Yeasts are essential for cocoa bean fermentation.

PubMed

Ho, Van Thi Thuy; Zhao, Jian; Fleet, Graham

2014-03-17

Cocoa beans (Theobroma cacao) are the major raw material for chocolate production and fermentation of the beans is essential for the development of chocolate flavor precursors. In this study, a novel approach was used to determine the role of yeasts in cocoa fermentation and their contribution to chocolate quality. Cocoa bean fermentations were conducted with the addition of 200ppm Natamycin to inhibit the growth of yeasts, and the resultant microbial ecology and metabolism, bean chemistry and chocolate quality were compared with those of normal (control) fermentations. The yeasts Hanseniaspora guilliermondii, Pichia kudriavzevii and Kluyveromyces marxianus, the lactic acid bacteria Lactobacillus plantarum and Lactobacillus fermentum and the acetic acid bacteria Acetobacter pasteurianus and Gluconobacter frateurii were the major species found in the control fermentation. In fermentations with the presence of Natamycin, the same bacterial species grew but yeast growth was inhibited. Physical and chemical analyses showed that beans fermented without yeasts had increased shell content, lower production of ethanol, higher alcohols and esters throughout fermentation and lesser presence of pyrazines in the roasted product. Quality tests revealed that beans fermented without yeasts were purplish-violet in color and not fully brown, and chocolate prepared from these beans tasted more acid and lacked characteristic chocolate flavor. Beans fermented with yeast growth were fully brown in color and gave chocolate with typical characters which were clearly preferred by sensory panels. Our findings demonstrate that yeast growth and activity were essential for cocoa bean fermentation and the development of chocolate characteristics. Crown Copyright © 2014. Published by Elsevier B.V. All rights reserved.

The role of tumour necrosis factor alpha and soluble tumour necrosis factor alpha receptors in the symptomatology of schizophrenia.

PubMed

Turhan, Levent; Batmaz, Sedat; Kocbiyik, Sibel; Soygur, Arif Haldun

2016-07-01

Background Immunological mechanisms may be responsible for the development and maintenance of schizophrenia symptoms. Aim The aim of this study is to measure tumour necrosis factor-alpha (TNF-α), soluble tumour necrosis factor-alpha receptor I (sTNF-αRI), and soluble tumour necrosis factor-alpha receptor II (sTNF-αRII) levels in patients with schizophrenia and healthy individuals, and to determine their relationship with the symptoms of schizophrenia. Methods Serum TNF-α, sTNF-αRI and sTNF-αRII levels were measured. The Positive and Negative Syndrome Scale (PANSS) was administered for patients with schizophrenia (n = 35), and the results were compared with healthy controls (n = 30). Hierarchical regression analyses were undertaken to predict the levels of TNF-α, sTNF-αRI and sTNF-αRII. Results No significant difference was observed in TNF-α levels, but sTNF-αRI and sTNF-αRII levels were lower in patients with schizophrenia. Serum sTNF-αRI and sTNF-αRII levels were found to be negatively correlated with the negative subscale score of the PANSS, and sTNF-αRI levels were also negatively correlated with the total score of the PANSS. Smoking, gender, body mass index were not correlated with TNF-α and sTNF-α receptor levels. Conclusions These results suggest that there may be a change in anti-inflammatory response in patients with schizophrenia due to sTNF-αRI and sTNF-αRII levels. The study also supports low levels of TNF activity in schizophrenia patients with negative symptoms.

Consideration of Real World Factors Influencing Greenhouse Gas Emissions in ALPHA

EPA Science Inventory

Discuss a variety of factors that influence the simulated fuel economy and GHG emissions that are often overlooked and updates made to ALPHA based on actual benchmarking data observed across a range of vehicles and transmissions. ALPHA model calibration is also examined, focusin...

Amino acids Thr56 and Thr58 are not essential for elongation factor 2 function in yeast.

PubMed

Bartish, Galyna; Moradi, Hossein; Nygård, Odd

2007-10-01

Yeast elongation factor 2 is an essential protein that contains two highly conserved threonine residues, T56 and T58, that could potentially be phosphorylated by the Rck2 kinase in response to environmental stress. The importance of residues T56 and T58 for elongation factor 2 function in yeast was studied using site directed mutagenesis and functional complementation. Mutations T56D, T56G, T56K, T56N and T56V resulted in nonfunctional elongation factor 2 whereas mutated factor carrying point mutations T56M, T56C, T56S, T58S and T58V was functional. Expression of mutants T56C, T56S and T58S was associated with reduced growth rate. The double mutants T56M/T58W and T56M/T58V were also functional but the latter mutant caused increased cell death and considerably reduced growth rate. The results suggest that the physiological role of T56 and T58 as phosphorylation targets is of little importance in yeast under standard growth conditions. Yeast cells expressing mutants T56C and T56S were less able to cope with environmental stress induced by increased growth temperatures. Similarly, cells expressing mutants T56M and T56M/T58W were less capable of adapting to increased osmolarity whereas cells expressing mutant T58V behaved normally. All mutants tested were retained their ability to bind to ribosomes in vivo. However, mutants T56D, T56G and T56K were under-represented on the ribosome, suggesting that these nonfunctional forms of elongation factor 2 were less capable of competing with wild-type elongation factor 2 in ribosome binding. The presence of nonfunctional but ribosome binding forms of elongation factor 2 did not affect the growth rate of yeast cells also expressing wild-type elongation factor 2.

Biotechnological production of carotenoids by yeasts: an overview

PubMed Central

2014-01-01

Nowadays, carotenoids are valuable molecules in different industries such as chemical, pharmaceutical, poultry, food and cosmetics. These pigments not only can act as vitamin A precursors, but also they have coloring and antioxidant properties, which have attracted the attention of the industries and researchers. The carotenoid production through chemical synthesis or extraction from plants is limited by low yields that results in high production costs. This leads to research of microbial production of carotenoids, as an alternative that has shown better yields than other aforementioned. In addition, the microbial production of carotenoids could be a better option about costs, looking for alternatives like the use of low-cost substrates as agro-industrials wastes. Yeasts have demonstrated to be carotenoid producer showing an important growing capacity in several agro-industrial wastes producing high levels of carotenoids. Agro-industrial wastes provide carbon and nitrogen source necessary, and others elements to carry out the microbial metabolism diminishing the production costs and avoiding pollution from these agro-industrial wastes to the environmental. Herein, we discuss the general and applied concepts regarding yeasts carotenoid production and the factors influencing carotenogenesis using agro-industrial wastes as low-cost substrates. PMID:24443802

Iodination of (Tyr11)somatostatin yields a super high affinity ligand for somatostatin receptors in GH4C1 pituitary cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Presky, D.H.; Schonbrunn, A.

1988-11-01

GH4C1 cells are a clonal strain of rat pituitary tumor cells which contain high affinity receptors for the inhibitory neuropeptide somatostatin (SRIF). In contrast to other peptides that bind to specific receptors on these cells, receptor-bound (125I-Tyr1)SRIF does not undergo rapid endocytosis. Rather, partial degradation to 125I-tyrosine occurs concomitantly with the dissociation of (125I-Tyr1)SRIF from cell surface receptors. In this study we characterize the binding, biological activity and receptor-mediated degradation of (125I-Tyr11)SRIF, a SRIF analog that is radiolabeled in the center of the molecule. The binding of trace concentrations of (125I-Tyr11)SRIF (less than 50 pM) required 6 hr to reachmore » equilibrium at 37 degrees compared with the 60 min required for (125I-Tyr1)SRIF. Analysis of the kinetics of (125I- Tyr11)SRIF binding showed that the rate constant for association (kon = 1.7 x 10(8) M-8min-1) was similar to that for (125I-Tyr1)SRIF (0.8 x 10(8) M-1min-1). However, the two radioligands exhibited markedly different dissociation kinetics; the koff for (125I-Tyr11)SRIF was 0.002 min-1 compared with the value of 0.02 min-1 for (125I-Tyr1) SRIF. In agreement with its much slower rate of dissociation, (125I-Tyr11)SRIF bound to the SRIF receptor with higher affinity (Kd = 70 pM) than did (125I-Tyr1)SRIF (Kd = 350 pM). However, the apparent ED50 for (I-Tyr11)SRIF to inhibit cAMP accumulation (1.9 +/- 0.4 nM) was greater than the ED50 for SRIF (0.19 +/- 0.04 nM). The low potency of (I-Tyr11)SRIF probably resulted from the fact that subsaturating concentrations of this peptide did not achieve equilibrium binding during the 30-min incubation used to assay biological activity. As previously reported for (125I-Tyr1)SRIF, receptor-bound (125I-Tyr11)SRIF was not internalized and was released from the cells as a mixture of intact (125I-Tyr11)SRIF (30%) and the degradation product 125I-tyrosine (65%).« less

In vitro biosynthesis of 17 alpha,20 alpha,20 beta-dihydroxy-4-pegnen-3-one by the ovaries, testes, and head kidneys of the Atlantic salmon Salmo salar

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sangalang, G.B.; Freeman, H.C.

Ovaries, testes, and head kidneys of sexually mature Atlantic salmon, Salmo salar, biosynthesized 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (17 alpha,20 beta-diOHP) from equimolar amounts of (/sup 3/H)pregnenolone plus (4-/sup 14/C)progesterone in vitro. The /sup 3/H:/sup 14/C isotope ratios of steroid metabolites indicated that the biosynthetic pathways to 17 alpha,20 beta-diOHP in the testes differed from those observed in the ovaries and head kidneys. (4-/sup 14/C)progesterone appeared to be the principal precursor of 17 alpha,20 beta-diOHP in the testes, whereas both precursors were efficiently biotransformed to 17 alpha,20 beta-diOPH in the ovaries and head kidneys. 17 alpha-Hydroxy-4-pregnen-3-one (17 alpha-OHP) was the immediate precursormore » to 17 alpha,20 beta-diOHP in all tissues. However, appreciable amounts of 17 alpha,20 beta-diOHP accumulated in vitro in the testes only in the presence of exogenous (/sup 14/C)progesterone. Incubation of the testes, ovaries, and head kidneys with (/sup 14/C)pregnenolone resulted in high yields of 17 alpha,20 beta-diOHP in the ovaries and head kidneys but no detectable amounts of the steroid in the testes. The results confirm that progesterone is the favored precursor to 17 alpha,20 beta-diOHP in the testes. The results also suggest that the head kidneys may be an excellent cellular source of 17 alpha,20 beta-diOHP in both male and female. Atlantic salmon and may play an important role in the sexual maturation process in this fish. It is suggested that biosynthetic control mechanism affecting 17 alpha,20 beta-diOHP synthesis and/or spermiation and ovulation may differ in male and female Atlantic salmon.« less

Parallel Inhibition of Dopamine Amacrine Cells and Intrinsically Photosensitive Retinal Ganglion Cells in a Non-Image-Forming Visual Circuit of the Mouse Retina

PubMed Central

Vuong, Helen E.; Hardi, Claudia N.; Barnes, Steven

2015-01-01

An inner retinal microcircuit composed of dopamine (DA)-containing amacrine cells and melanopsin-containing, intrinsically photosensitive retinal ganglion cells (M1 ipRGCs) process information about the duration and intensity of light exposures, mediating light adaptation, circadian entrainment, pupillary reflexes, and other aspects of non-image-forming vision. The neural interaction is reciprocal: M1 ipRGCs excite DA amacrine cells, and these, in turn, feed inhibition back onto M1 ipRGCs. We found that the neuropeptide somatostatin [somatotropin release inhibiting factor (SRIF)] also inhibits the intrinsic light response of M1 ipRGCs and postulated that, to tune the bidirectional interaction of M1 ipRGCs and DA amacrine cells, SRIF amacrine cells would provide inhibitory modulation to both cell types. SRIF amacrine cells, DA amacrine cells, and M1 ipRGCs form numerous contacts. DA amacrine cells and M1 ipRGCs express the SRIF receptor subtypes sst2A and sst4 respectively. SRIF modulation of the microcircuit was investigated with targeted patch-clamp recordings of DA amacrine cells in TH–RFP mice and M1 ipRGCs in OPN4–EGFP mice. SRIF increases K+ currents, decreases Ca2+ currents, and inhibits spike activity in both cell types, actions reproduced by the selective sst2A agonist L-054,264 (N-[(1R)-2-[[[(1S*,3R*)-3-(aminomethyl)cyclohexyl]methyl]amino]-1-(1H-indol-3-ylmethyl)-2-oxoethyl]spiro[1H-indene-1,4′-piperidine]-1′-carboxamide) in DA amacrine cells and the selective sst4 agonist L-803,087 (N2-[4-(5,7-difluoro-2-phenyl-1H-indol-3-yl)-1-oxobutyl]-l-arginine methyl ester trifluoroacetate) in M1 ipRGCs. These parallel actions of SRIF may serve to counteract the disinhibition of M1 ipRGCs caused by SRIF inhibition of DA amacrine cells. This allows the actions of SRIF on DA amacrine cells to proceed with adjusting retinal DA levels without destabilizing light responses by M1 ipRGCs, which project to non-image-forming targets in the brain. SIGNIFICANCE

Identification of novel host factors via conserved domain search: Cns1 cochaperone is a novel restriction factor of tombusvirus replication in yeast.

PubMed

Lin, Jing-Yi; Nagy, Peter D

2013-12-01

A large number of host-encoded proteins affect the replication of plus-stranded RNA viruses by acting as susceptibility factors. Many other cellular proteins are known to function as restriction factors of viral infections. Previous studies with tomato bushy stunt tombusvirus (TBSV) in a yeast model host have revealed the inhibitory function of TPR (tetratricopeptide repeat) domain-containing cyclophilins, which are members of the large family of host prolyl isomerases, in TBSV replication. In this paper, we tested additional TPR-containing yeast proteins in a cell-free TBSV replication assay and identified the Cns1p cochaperone for heat shock protein 70 (Hsp70) and Hsp90 chaperones as a strong inhibitor of TBSV replication. Cns1p interacted with the viral replication proteins and inhibited the assembly of the viral replicase complex and viral RNA synthesis in vitro. Overexpression of Cns1p inhibited TBSV replication in yeast. The use of a temperature-sensitive (TS) mutant of Cns1p in yeast revealed that at a semipermissive temperature, TS Cns1p could not inhibit TBSV replication. Interestingly, Cns1p and the TPR-containing Cpr7p cyclophilin have similar inhibitory functions during TBSV replication, although some of the details of their viral restriction mechanisms are different. Our observations indicate that TPR-containing cellular proteins could act as virus restriction factors.

Development of antibodies against the rat brain somatostatin receptor.

PubMed

Theveniau, M; Rens-Domiano, S; Law, S F; Rougon, G; Reisine, T

1992-05-15

Somatostatin (SRIF) is a neurotransmitter in the brain involved in the regulation of motor activity and cognition. It induces its physiological actions by interacting with receptors. We have developed antibodies against the receptor to investigate its structural properties. Rabbit polyclonal antibodies were generated against the rat brain SRIF receptor. These antibodies (F4) were able to immunoprecipitate solubilized SRIF receptors from rat brain and the cell line AtT-20. The specificity of the interaction of these antibodies with SRIF receptors was further demonstrated by immunoblotting. F4 detected SRIF receptors of 60 kDa from rat brain and adrenal cortex and the cell lines AtT-20, GH3, and NG-108, which express high densities of SRIF receptors. They did not detect immunoreactive material from rat liver or COS-1, HEPG, or CRL cells, which do not express functional SRIF receptors. In rat brain, 60-kDa immunoreactivity was detected by F4 in the hippocampus, cerebral cortex, and striatum, which have high densities of SRIF receptors. However, F4 did not interact with proteins from cerebellum and brain stem, which express few SRIF receptors. Immunoreactive material cannot be detected in rat pancreas or pituitary, which have been reported to express a 90-kDa SRIF receptor subtype. The selective detection of 60-kDa SRIF receptors by F4 indicates that the 60- and 90-kDa SRIF receptor subtypes are immunologically distinct. The availability of antibodies that selectively detect native and denatured brain SRIF receptors provides us with a feasible approach to clone the brain SRIF receptor gene(s).

Orange-spotted grouper (Epinephelus coioides) orexin: molecular cloning, tissue expression, ontogeny, daily rhythm and regulation of NPY gene expression.

PubMed

Yan, Aifen; Zhang, Lingjuang; Tang, Zhiguo; Zhang, Yanhong; Qin, Chaobin; Li, Bo; Li, Wensheng; Lin, Haoran

2011-07-01

Orexin-A and -B, collectively called orexins, are hypothalamic neuropeptides involved in the regulation of food intake, sleep and energy balance. In this study, the full-length cDNA of prepro-orexin was isolated from the hypothalamus of orange-spotted grouper (Epinephelus coioides) using RT-PCR and RACE. The grouper prepro-orexin cDNA is 711 bp in length and encodes a 149-amino acid precursor protein that contains a 46-amino acid signal peptide, a 43-amino acid mature orexin-A peptide, a 27-amino acid mature orexin-B peptide and a 33-amino acid C terminus of unknown function. The tissue distribution and ontogeny of prepro-orexin were examined by quantitative real-time PCR. We found that the prepro-orexin mRNA is widely expressed in brain and peripheral tissues, with abundant expression in the hypothalamus. During the embryonic development, prepro-orexin mRNA was first detected in neurula stage embryos, and its expression gradually increased during the remainder of embryogenesis. Our analysis of grouper hypothalamic prepro-orexin expression showed that prepro-orexin mRNA levels were greater in the light phase than in the dark phase and increased significantly at meal-time. Intraperitoneal injection of orexin-A caused a dose-related increase in hypothalamus NPY mRNA expression level after 4h. Orexin-A also increased NPY mRNA expression level from static hypothalamic fragments incubation. Our results imply that orexin may be involved in feeding in the orange-spotted grouper and orexin-A is a stimulator of NPY mRNA expression in vivo and in vitro. Copyright © 2011 Elsevier Inc. All rights reserved.

alpha-Galactosidase from Bacillus megaterium VHM1 and its application in removal of flatulence-causing factors from soymilk.

PubMed

Patil, Aravind Goud G; K, Praveen Kumar S; Mulimani, Veerappa H; Veeranagouda, Yaligara; Lee, Kyoung

2010-11-01

A bacterial strain capable of producing extracellular alpha-galactosidase was isolated from sugar cane industrial waste soil sample. Microbiological, physiological, and biochemical studies revealed that isolate belonged to Bacillus sp,. Furthermore, 16S rDNA sequence analysis of new isolates was identified as Bacillus megaterium VHM1. The production of alpha-galactosidase was optimized by various physical culture conditions. Guar gum and yeast extract acted as the best carbon and nitrogen source, respectively for the production of alpha-galactosidase. The enzyme showed an optimum pH at 7.5 and was stable over a pH between 5 and 9. The enzyme was optimally active in 55degreesC and the enzyme was thermostable with half life of 120 minutes at 55 degrees C and lost their 90%, residual activity in 120 minutes at 60 degrees C. alpha-Galactosidase was strongly inhibited by Ag2, Cu2, and Hg2+ at 1mM concentration. The metal ions Fe2, Mn2+, and Mg2+ had no effect on alpha-galactosidase activity, Zn2+,Ni2+, and Ca2+ reduced the enzyme activity slightly. The B megaterium VHM1 enzyme treatment completely hydrolyzed flatulence-causing sugars of soymilk within one and half hour.

Chiral Polyfunctional Thiols and Their Conjugated Precursors upon Winemaking with Five Vitis vinifera Sauvignon blanc Clones.

PubMed

Chen, Liang; Capone, Dimitra L; Tondini, Federico A; Jeffery, David W

2018-05-09

Five co-located clones of Sauvignon blanc grapes were fermented under controlled conditions at laboratory-scale to investigate the impact of yeast strain, commercial enzyme, or nutrient addition on the concentrations of enantiomers of 3-sulfanylhexan-1-ol (3-SH) and 3-sulfanylhexyl acetate (3-SHA) in resulting wines. The relationship of these enantiomers with the odorless 3-SH precursors present in diastereomeric forms in grape juice was also examined. Possible variations may have existed due to clone type, not only for the diastereomers of 3-SH precursors in juices but also for the enantiomers of 3-SH and 3-SHA in the resulting wines, although there was no obvious stereochemical relationship between precursors and free thiols. From a flavor enhancement perspective, the use of a commercial enzyme in the juice significantly enhanced 3-SH production for some clones. In contrast, less impact on the production of 3-SH and 3-SHA was seen as a result of yeast strain and nutrient regardless of clone type.

Dedifferentiated Schwann Cell Precursors Secreting Paracrine Factors Are Required for Regeneration of the Mammalian Digit Tip.

PubMed

Johnston, Adam P W; Yuzwa, Scott A; Carr, Matthew J; Mahmud, Neemat; Storer, Mekayla A; Krause, Matthew P; Jones, Karen; Paul, Smitha; Kaplan, David R; Miller, Freda D

2016-10-06

Adult mammals have lost multi-tissue regenerative capacity, except for the distal digit, which is able to regenerate via mechanisms that remain largely unknown. Here, we show that, after adult mouse distal digit removal, nerve-associated Schwann cell precursors (SCPs) dedifferentiate and secrete growth factors that promote expansion of the blastema and digit regeneration. When SCPs were dysregulated or ablated, mesenchymal precursor proliferation in the blastema was decreased and nail and bone regeneration were impaired. Transplantation of exogenous SCPs rescued these regeneration defects. We found that SCPs secrete factors that promote self-renewal of mesenchymal precursors, and we used transcriptomic and proteomic analysis to define candidate factors. Two of these, oncostatin M (OSM) and platelet-derived growth factor AA (PDGF-AA), are made by SCPs in the regenerating digit and rescued the deficits in regeneration caused by loss of SCPs. As all peripheral tissues contain nerves, these results could have broad implications for mammalian tissue repair and regeneration. Copyright © 2016 Elsevier Inc. All rights reserved.

Expression of hypoxia-induced factor-1 alpha in early-stage and in metastatic oral squamous cell carcinoma.

PubMed

Ribeiro, Maisa; Teixeira, Sarah R; Azevedo, Monarko N; Fraga, Ailton C; Gontijo, Antônio Pm; Vêncio, Eneida F

2017-04-01

To investigate hypoxia-induced factor-1 alpha expression in distinct oral squamous cell carcinoma subtypes and topographies and correlate with clinicopathological data. Hypoxia-induced factor-1 alpha expression was assessed by immunohistochemistry in 93 cases of OSCC. Clinical and histopathological data were reviewed from medical records. Hypoxia-induced factor-1 alpha status was distinct according to tumor location, subtype and topography affect. In superficial oral squamous cell carcinomas, most tumor cells overexpressed hypoxia-induced factor-1 alpha, whereas hypoxia-induced factor-1 alpha was restricted to the intratumoral region in conventional squamous cell carcinomas. All basaloid squamous cell carcinomas exhibited downregulation of hypoxia-induced factor-1 alpha. Interestingly, metastatic lymph nodes (91.7%, p = 0.001) and the intratumoral regions of corresponding primary tumors (58.3%, p = 0.142) showed hypoxia-induced factor-1 alpha-positive tumor cells. Overall survival was poor in patients with metastatic lymph nodes. Hypoxia-induced factor-1 alpha has distinct expression patterns in different oral squamous cell carcinoma subtypes and topographies, suggesting that low oxygen tension promotes the growth pattern of superficial and conventional squamous cell carcinoma, but not basaloid squamous cell carcinoma. Indeed, a hypoxic environment may facilitate regional metastasis, making it a useful diagnostic and prognostic marker in primary tumors.

Advances in synthetic biology of oleaginous yeast Yarrowia lipolytica for producing non-native chemicals.

PubMed

Darvishi, Farshad; Ariana, Mehdi; Marella, Eko Roy; Borodina, Irina

2018-07-01

Oleaginous yeast Yarrowia lipolytica is an important industrial host for the production of enzymes, oils, fragrances, surfactants, cosmetics, and pharmaceuticals. More recently, improved synthetic biology tools have allowed more extensive engineering of this yeast species, which lead to the production of non-native metabolites. In this review, we summarize the recent advances of genome editing tools for Y. lipolytica, including the application of CRISPR/Cas9 system and discuss case studies, where Y. lipolytica was engineered to produce various non-native chemicals: short-chain fatty alcohols and alkanes as biofuels, polyunsaturated fatty acids for nutritional and pharmaceutical applications, polyhydroxyalkanoates and dicarboxylic acids as precursors for biodegradable plastics, carotenoid-type pigments for food and feed, and campesterol as a precursor for steroid drugs.

Induction of human airway hyperresponsiveness by tumour necrosis factor-alpha.

PubMed

Anticevich, S Z; Hughes, J M; Black, J L; Armour, C L

1995-09-15

Tumour necrosis factor-alpha (TNF alpha) is implicated in the pathogenesis of asthma; however, little is known of its direct effect on smooth muscle reactivity. We investigated the effect of TNF alpha on the responsiveness of human bronchial tissue to electrical field stimulation in vitro. Incubation of non-sensitized tissue with 1 nM, 3 nM and 10 nM TNF alpha significantly increased responsiveness to electrical field stimulation (113 +/- 8, 110 +/- 4 and 112 +/- 2% respectively) compared to control (99 +/- 2%) (P < 0.05, n = 6). Responses were not increased in sensitized tissue (101 +/- 3% versus 105 +/- 5%, n = 3, P > 0.05) nor were responses to exogenous acetylcholine (93 +/- 4% versus 73 +/- 7%, n = 3, P = 0.38). These results show that TNF alpha causes an increase in responsiveness of human bronchial tissue and that this occurs prejunctionally on the parasympathetic nerve pathway. This is the first report of a cytokine increasing human airway tissue responsiveness.

The Fermentative and Aromatic Ability of Kloeckera and Hanseniaspora Yeasts

NASA Astrophysics Data System (ADS)

Díaz-Montaño, Dulce M.; de Jesús Ramírez Córdova, J.

Spontaneous alcoholic fermentation from grape, agave and others musts into an alcoholic beverage is usually characterized by the presence of several non-Saccharomyces yeasts. These genera yeasts are dominant in the early stages of the alcoholic fermentation. However the genera Hanseniaspora and Kloeckera may survive at a significant level during fermentation and can influence the chemical composition of the beverage. Several strains belonging to the species Kloeckera api-culata and Hanseniaspora guilliermondii have been extensively studied in relation to the formation of some metabolic compounds affecting the bouquet of the final product. Indeed some apiculate yeast showed positive oenological properties and their use in the alcoholic fermentations has been suggested to enhance the aroma and flavor profiles. The non- Saccharomyces yeasts have the capability to produce and secrete enzymes in the medium, such as β -glucosidases, which release monoterpenes derived from their glycosylated form. These compounds contribute to the higher fruit-like characteristic of final product. This chapter reviews metabolic activity of Kloeckera and Hanseniaspora yeasts in several aspects: fermentative capability, aromatic compounds production and transformation of aromatic precursor present in the must, also covers the molecular methods for identifying of the yeast

The hydrolytic activity of esterases in the yeast Saccharomyces cerevisiae is strain dependent.

PubMed

Kwolek-Mirek, Magdalena; Bednarska, Sabina; Zadrąg-Tęcza, Renata; Bartosz, Grzegorz

2011-11-01

Ester precursors of fluorogenic or chromogenic probes are often employed in studies of yeast cell biology. This study was aimed at a comparison of the ability of several commonly used laboratory wild-type Saccharomyces cerevisiae strains to hydrolyse the following model esters: fluorescein diacetate, 2-naphthyl acetate, PNPA (p-nitrophenyl acetate) and AMQI (7-acetoxy-1-methylquinolinum iodide). In all the strains, the esterase activity was localized mainly to the cytosol. Considerable differences in esterase activity were observed between various wild-type laboratory yeast strains. The phase of growth also contributed to the variation in esterase activity of the yeast. This diversity implies the need for caution in using intracellularly hydrolysed probes for a comparison of yeast strains with various genetic backgrounds.

A serendipitous discovery that in situ proteolysis is essential for the crystallization of yeast CPSF-100 (Ydh1p)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mandel, Corey R.; Gebauer, Damara; Zhang, Hailong

2006-10-01

Proteolysis in situ by a protease secreted by a contaminating fungus is essential for the crystallization of yeast CPSF-100. The cleavage and polyadenylation specificity factor (CPSF) complex is required for the cleavage and polyadenylation of the 3′-end of messenger RNA precursors in eukaryotes. During structural studies of the 100 kDa subunit (CPSF-100, Ydh1p) of the yeast CPSF complex, it was serendipitously discovered that a solution that is infected by a fungus (subsequently identified as Penicillium) is crucial for the crystallization of this protein. Further analyses suggest that the protein has undergone partial proteolysis during crystallization, resulting in the deletion ofmore » an internal segment of about 200 highly charged and hydrophilic residues, very likely catalyzed by a protease secreted by the fungus. With the removal of this segment, yeast CPSF-100 (Ydh1p) has greatly reduced solubility and can be crystallized in the presence of a minute amount of precipitant.« less

The Gcn4 transcription factor reduces protein synthesis capacity and extends yeast lifespan.

PubMed

Mittal, Nitish; Guimaraes, Joao C; Gross, Thomas; Schmidt, Alexander; Vina-Vilaseca, Arnau; Nedialkova, Danny D; Aeschimann, Florian; Leidel, Sebastian A; Spang, Anne; Zavolan, Mihaela

2017-09-06

In Saccharomyces cerevisiae, deletion of large ribosomal subunit protein-encoding genes increases the replicative lifespan in a Gcn4-dependent manner. However, how Gcn4, a key transcriptional activator of amino acid biosynthesis genes, increases lifespan, is unknown. Here we show that Gcn4 acts as a repressor of protein synthesis. By analyzing the messenger RNA and protein abundance, ribosome occupancy and protein synthesis rate in various yeast strains, we demonstrate that Gcn4 is sufficient to reduce protein synthesis and increase yeast lifespan. Chromatin immunoprecipitation reveals Gcn4 binding not only at genes that are activated, but also at genes, some encoding ribosomal proteins, that are repressed upon Gcn4 overexpression. The promoters of repressed genes contain Rap1 binding motifs. Our data suggest that Gcn4 is a central regulator of protein synthesis under multiple perturbations, including ribosomal protein gene deletions, calorie restriction, and rapamycin treatment, and provide an explanation for its role in longevity and stress response.The transcription factor Gcn4 is known to regulate yeast amino acid synthesis. Here, the authors show that Gcn4 also acts as a repressor of protein biosynthesis in a range of conditions that enhance yeast lifespan, such as ribosomal protein knockout, calorie restriction or mTOR inhibition.

Glutathione regulation of redox-sensitive signals in tumor necrosis factor-{alpha}-induced vascular endothelial dysfunction

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tsou, T.-C.; Yeh, S.C.; Tsai, F.-Y.

2007-06-01

We investigated the regulatory role of glutathione in tumor necrosis factor-alpha (TNF-{alpha})-induced vascular endothelial dysfunction as evaluated by using vascular endothelial adhesion molecule expression and monocyte-endothelial monolayer binding. Since TNF-{alpha} induces various biological effects on vascular cells, TNF-{alpha} dosage could be a determinant factor directing vascular cells into different biological fates. Based on the adhesion molecule expression patterns responding to different TNF-{alpha} concentrations, we adopted the lower TNF-{alpha} (0.2 ng/ml) to rule out the possible involvement of other TNF-{alpha}-induced biological effects. Inhibition of glutathione synthesis by L-buthionine-(S,R)-sulfoximine (BSO) resulted in down-regulations of the TNF-{alpha}-induced adhesion molecule expression and monocyte-endothelial monolayermore » binding. BSO attenuated the TNF-{alpha}-induced nuclear factor-kappaB (NF-{kappa}B) activation, however, with no detectable effect on AP-1 and its related mitogen-activated protein kinases (MAPKs). Deletion of an AP-1 binding site in intercellular adhesion molecule-1 (ICAM-1) promoter totally abolished its constitutive promoter activity and its responsiveness to TNF-{alpha}. Inhibition of ERK, JNK, or NF-{kappa}B attenuates TNF-{alpha}-induced ICAM-1 promoter activation and monocyte-endothelial monolayer binding. Our study indicates that TNF-{alpha} induces adhesion molecule expression and monocyte-endothelial monolayer binding mainly via activation of NF-{kappa}B in a glutathione-sensitive manner. We also demonstrated that intracellular glutathione does not modulate the activation of MAPKs and/or their downstream AP-1 induced by lower TNF-{alpha}. Although AP-1 activation by the lower TNF-{alpha} was not detected in our systems, we could not rule out the possible involvement of transiently activated MAPKs/AP-1 in the regulation of TNF-{alpha}-induced adhesion molecule expression.« less

An Exploratory Study of Alpha Contracting: Antecedents, Processes, Issues, Success Factors and Consequences

DTIC Science & Technology

2009-09-01

ABSTRACT (maximum 200 words) Alpha contracting is a collaborative effort between a buyer and supplier during contract formation to maximize efficiency...experienced Alpha contracting teams, to include contracting officers, DCAA, DCMA, end users/ customers , program managers and acquisition directors to better...PROCESSES, ISSUES, SUCCESS FACTORS AND CONSEQUENCES ABSTRACT Alpha contracting is a collaborative effort between a buyer and supplier

Identification of the Transcription Factor Znc1p, which Regulates the Yeast-to-Hypha Transition in the Dimorphic Yeast Yarrowia lipolytica

PubMed Central

Martinez-Vazquez, Azul; Gonzalez-Hernandez, Angelica; Domínguez, Ángel; Rachubinski, Richard; Riquelme, Meritxell; Cuellar-Mata, Patricia; Guzman, Juan Carlos Torres

2013-01-01

The dimorphic yeast Yarrowia lipolytica is used as a model to study fungal differentiation because it grows as yeast-like cells or forms hyphal cells in response to changes in environmental conditions. Here, we report the isolation and characterization of a gene, ZNC1, involved in the dimorphic transition in Y. lipolytica. The ZNC1 gene encodes a 782 amino acid protein that contains a Zn(II)2C6 fungal-type zinc finger DNA-binding domain and a leucine zipper domain. ZNC1 transcription is elevated during yeast growth and decreases during the formation of mycelium. Cells in which ZNC1 has been deleted show increased hyphal cell formation. Znc1p-GFP localizes to the nucleus, but mutations within the leucine zipper domain of Znc1p, and to a lesser extent within the Zn(II)2C6 domain, result in a mislocalization of Znc1p to the cytoplasm. Microarrays comparing gene expression between znc1::URA3 and wild-type cells during both exponential growth and the induction of the yeast-to-hypha transition revealed 1,214 genes whose expression was changed by 2-fold or more under at least one of the conditions analyzed. Our results suggest that Znc1p acts as a transcription factor repressing hyphal cell formation and functions as part of a complex network regulating mycelial growth in Y. lipolytica. PMID:23826133

Nrt1 and Tna1-independent export of NAD+ precursor vitamins promotes NAD+ homeostasis and allows engineering of vitamin production.

PubMed

Belenky, Peter; Stebbins, Rebecca; Bogan, Katrina L; Evans, Charles R; Brenner, Charles

2011-05-11

NAD(+) is both a co-enzyme for hydride transfer enzymes and a substrate of sirtuins and other NAD(+) consuming enzymes. NAD(+) biosynthesis is required for two different regimens that extend lifespan in yeast. NAD(+) is synthesized from tryptophan and the three vitamin precursors of NAD(+): nicotinic acid, nicotinamide and nicotinamide riboside. Supplementation of yeast cells with NAD(+) precursors increases intracellular NAD(+) levels and extends replicative lifespan. Here we show that both nicotinamide riboside and nicotinic acid are not only vitamins but are also exported metabolites. We found that the deletion of the nicotinamide riboside transporter, Nrt1, leads to increased export of nicotinamide riboside. This discovery was exploited to engineer a strain to produce high levels of extracellular nicotinamide riboside, which was recovered in purified form. We further demonstrate that extracellular nicotinamide is readily converted to extracellular nicotinic acid in a manner that requires intracellular nicotinamidase activity. Like nicotinamide riboside, export of nicotinic acid is elevated by the deletion of the nicotinic acid transporter, Tna1. The data indicate that NAD(+) metabolism has a critical extracellular element in the yeast system and suggest that cells regulate intracellular NAD(+) metabolism by balancing import and export of NAD(+) precursor vitamins.

Acetylcholine Modulates the Hormones of the Growth Hormone/Insulinlike Growth Factor-1 Axis During Development in Mice.

PubMed

Lecomte, Marie-José; Bertolus, Chloé; Ramanantsoa, Nélina; Saurini, Françoise; Callebert, Jacques; Sénamaud-Beaufort, Catherine; Ringot, Maud; Bourgeois, Thomas; Matrot, Boris; Collet, Corinne; Nardelli, Jeannette; Mallet, Jacques; Vodjdani, Guilan; Gallego, Jorge; Launay, Jean-Marie; Berrard, Sylvie

2018-04-01

Pituitary growth hormone (GH) and insulinlike growth factor (IGF)-1 are anabolic hormones whose physiological roles are particularly important during development. The activity of the GH/IGF-1 axis is controlled by complex neuroendocrine systems including two hypothalamic neuropeptides, GH-releasing hormone (GHRH) and somatostatin (SRIF), and a gastrointestinal hormone, ghrelin. The neurotransmitter acetylcholine (ACh) is involved in tuning GH secretion, and its GH-stimulatory action has mainly been shown in adults but is not clearly documented during development. ACh, together with these hormones and their receptors, is expressed before birth, and somatotroph cells are already responsive to GHRH, SRIF, and ghrelin. We thus hypothesized that ACh could contribute to the modulation of the main components of the somatotropic axis during development. In this study, we generated a choline acetyltransferase knockout mouse line and showed that heterozygous mice display a transient deficit in ACh from embryonic day 18.5 to postnatal day 10, and they recover normal ACh levels from the second postnatal week. This developmental ACh deficiency had no major impact on weight gain and cardiorespiratory status of newborn mice. Using this mouse model, we found that endogenous ACh levels determined the concentrations of circulating GH and IGF-1 at embryonic and postnatal stages. In particular, serum GH level was correlated with brain ACh content. ACh also modulated the levels of GHRH and SRIF in the hypothalamus and ghrelin in the stomach, and it affected the levels of these hormones in the circulation. This study identifies ACh as a potential regulator of the somatotropic axis during the developmental period.

Stereospecific deuteration of alpha-furanosyl azomycin nucleosides: a model reaction for tritium radiolabeling.

PubMed

Kumar, Piyush; Emami, Saeed; McEwan, Alexander J B; Wiebe, Leonard I

2008-06-01

Stereospecific synthesis of 1-alpha-d-(2-deuteroribofuranosyl)-2-nitroimidazole (2'-[(2)H]-alpha-AZR) is reported. This, deuteration was independent of the configuration of C-2' -OH group (arabinose or ribose) in sugar moiety of starting molecules. Slightly better yield (>37%) of the deuterated product, 6, from arabinosyl precursor in comparison to corresponding ribose precursor (29%) was obtained which may reflect better stereochemical availability of C-2' -OH in arabinose during oxidation.

Cyclo(dehydroala-L-Leu), an alpha-glucosidase inhibitor from Penicillium sp. F70614.

PubMed

Kwon, O S; Park, S H; Yun, B S; Pyun, Y R; Kim, C J

2000-09-01

A diketopiperazine (1) has been isolated from the culture broth of Penicillium sp. F70614 and its structure has been determined to be cyclo(dehydroala-L-Leu) by various spectroscopic analyses. This compound selectively inhibited yeast alpha-glucosidase and porcine intestinal alpha-glucosidase with IC50 values of 35 and 50 microg/ml, respectively. However, it did not show significant inhibitory effects against almond beta3-glucosidase, Aspergillus alpha-galactosidase, Escherichia coli beta-galactosidase and jack bean alpha-mannosidase.

Sec63p and Kar2p are required for the translocation of SRP-dependent precursors into the yeast endoplasmic reticulum in vivo

PubMed Central

Young, Barry P.; Craven, Rachel A.; Reid, Peter J.; Willer, Martin; Stirling, Colin J.

2001-01-01

The translocation of secretory polypeptides into the endoplasmic reticulum (ER) occurs at the translocon, a pore-forming structure that orchestrates the transport and maturation of polypeptides at the ER membrane. In yeast, targeting of secretory precursors to the translocon can occur by two distinct pathways that are distinguished by their dependence upon the signal recognition particle (SRP). The SRP-dependent pathway requires SRP and its membrane-bound receptor, whereas the SRP-independent pathway requires a separate receptor complex consisting of Sec62p, Sec63p, Sec71p, Sec72p plus lumenal Kar2p/BiP. Here we demonstrate that Sec63p and Kar2p are also required for the SRP-dependent targeting pathway in vivo. Furthermore, we demonstrate multiple roles for Sec63p, at least one of which is exclusive to the SRP-independent pathway. PMID:11226176

Hantaan virus nucleocapsid protein binds to importin alpha proteins and inhibits tumor necrosis factor alpha-induced activation of nuclear factor kappa B.

PubMed

Taylor, Shannon L; Frias-Staheli, Natalia; García-Sastre, Adolfo; Schmaljohn, Connie S

2009-02-01

Hantaviruses such as Hantaan virus (HTNV) and Andes virus cause two human diseases, hemorrhagic fever with renal syndrome and hantavirus pulmonary syndrome, respectively. For both, disease pathogenesis is thought to be immunologically mediated and there have been numerous reports of patients with elevated levels of proinflammatory and inflammatory cytokines, including tumor necrosis factor alpha (TNF-alpha), in their sera. Multiple viruses have developed evasion strategies to circumvent the host cell inflammatory process, with one of the most prevalent being the disruption of nuclear factor kappa B (NF-kappaB) activation. We hypothesized that hantaviruses might also moderate host inflammation by interfering with this pathway. We report here that the nucleocapsid (N) protein of HTNV was able to inhibit TNF-alpha-induced activation of NF-kappaB, as measured by a reporter assay, and the activation of endogenous p65, an NF-kappaB subunit. Surprisingly, there was no defect in the degradation of the inhibitor of NF-kappaB (IkappaB) protein, nor was there any alteration in the level of p65 expression in HTNV N-expressing cells. However, immunofluorescence antibody staining demonstrated that cells expressing HTNV N protein and a green fluorescent protein-p65 fusion had limited p65 nuclear translocation. Furthermore, we were able to detect an interaction between HTNV N protein and importin alpha, a nuclear import molecule responsible for shuttling NF-kappaB to the nucleus. Collectively, our data suggest that HTNV N protein can sequester NF-kappaB in the cytoplasm, thus inhibiting NF-kappaB activity. These findings, which were obtained using cells transfected with cDNA representing the HTNV N gene, were confirmed using HTNV-infected cells.

Combinations of ERK and p38 MAPK inhibitors ablate tumor necrosis factor-alpha (TNF-alpha ) mRNA induction. Evidence for selective destabilization of TNF-alpha transcripts.

PubMed

Rutault, K; Hazzalin, C A; Mahadevan, L C

2001-03-02

Tumor necrosis factor-alpha (TNF-alpha) is a potent proinflammatory cytokine whose synthesis and secretion are implicated in diverse pathologies. Hence, inhibition of TNF-alpha transcription or translation and neutralization of its protein product represent major pharmaceutical strategies to control inflammation. We have studied the role of ERK and p38 mitogen-activated protein (MAP) kinase in controlling TNF-alpha mRNA levels in differentiated THP-1 cells and in freshly purified human monocytes. We show here that it is possible to produce virtually complete inhibition of lipopolysaccharide-stimulated TNF-alpha mRNA accumulation by using a combination of ERK and p38 MAP kinase inhibitors. Furthermore, substantial inhibition is achievable using combinations of 1 microm of each inhibitor, whereas inhibitors used individually are incapable of producing complete inhibition even at high concentrations. Finally, addressing mechanisms involved, we show that inhibition of p38 MAP kinase selectively destabilizes TNF-alpha transcripts but does not affect degradation of c-jun transcripts. These results impinge on the controversy in the literature surrounding the mode of action of MAP kinase inhibitors on TNF-alpha mRNA and suggest the use of combinations of MAP kinase inhibitors as an effective anti-inflammatory strategy.

Plant-Derived Transcription Factors for Orthologous Regulation of Gene Expression in the Yeast Saccharomyces cerevisiae.

PubMed

Naseri, Gita; Balazadeh, Salma; Machens, Fabian; Kamranfar, Iman; Messerschmidt, Katrin; Mueller-Roeber, Bernd

2017-09-15

Control of gene expression by transcription factors (TFs) is central in many synthetic biology projects for which a tailored expression of one or multiple genes is often needed. As TFs from evolutionary distant organisms are unlikely to affect gene expression in a host of choice, they represent excellent candidates for establishing orthogonal control systems. To establish orthogonal regulators for use in yeast (Saccharomyces cerevisiae), we chose TFs from the plant Arabidopsis thaliana. We established a library of 106 different combinations of chromosomally integrated TFs, activation domains (yeast GAL4 AD, herpes simplex virus VP64, and plant EDLL) and synthetic promoters harboring cognate cis-regulatory motifs driving a yEGFP reporter. Transcriptional output of the different driver/reporter combinations varied over a wide spectrum, with EDLL being a considerably stronger transcription activation domain in yeast than the GAL4 activation domain, in particular when fused to Arabidopsis NAC TFs. Notably, the strength of several NAC-EDLL fusions exceeded that of the strong yeast TDH3 promoter by 6- to 10-fold. We furthermore show that plant TFs can be used to build regulatory systems encoded by centromeric or episomal plasmids. Our library of TF-DNA binding site combinations offers an excellent tool for diverse synthetic biology applications in yeast.

Effects of transforming growth factor-alpha (TGF-alpha) in vitro and in vivo on reovirus replication.

PubMed

Organ, Edward L; Nalbantyan, Christopher D; Nanney, Lillian B; Woodward, Stephen C; Sheng, Jinsong; Dubois, Raymond N; Price, James; Sutcliffe, Marilyn; Coffey, Robert J; Rubin, Donald H

2004-07-01

We have utilized growth factors in in vitro and in vivo systems to examine the role of cellular proliferation in reovirus replication. In vitro, proliferating RIE-1 cells can be infected with whole reovirus virions, but are relatively resistant to infection once confluent (Go arrest). It has been shown that TGF-alpha, which signals through the EGF-receptor (EGF-R), is capable of dramatically increasing the number of RIE-1 cells entering the S-phase in the presence of additional serum factors. Stimulation of the EGF-R without serum results in minimal increases in cells entering the S-phase with a restriction in reovirus replication. Therefore, other factors in serum are essential for fully permissive infection. In vivo, we used metallothionein (MT) promoter/enhancer-TGF-alpha transgenic mice to study the effect of cytokine activation on reovirus type 1 infection. Virus replication decreased following oral infection in these transgenic mice at 1 month of age, concordant with increased mucin production. Titers of reovirus obtained from the livers of 1 year old transgenic mice were approximately 10-fold higher than titers obtained in control mice. Taken together, these data indicate that while growth factor activation ultimately leads to an increase in virus infectivity, other factors may be necessary for reovirus replication.

Antarctic Yeasts: Biodiversity and Potential Applications

NASA Astrophysics Data System (ADS)

Shivaji, S.; Prasad, G. S.

This review is an attempt in cataloguing the diversity of yeasts in Antarctica, highlight their biotechnological potential and understand the basis of adaptation to low temperature. As of now several psychrophilic and psychrotolerant yeasts from Antarctic soils and marine waters have been characterized with respect to their growth characteristics, ecological distribution and taxonomic significance. Interestingly most of these species belonged to basidiomycetous yeasts which as a group are known for their ability to circumvent and survive under stress conditions. Simultaneously their possible role as work horses in the biotechnological industry was recognized due to their ability to produce novel enzymes and biomolecules such as agents for the breakdown of xenobiotics, and novel pharmaceutical chemi cals. The high activity of psychrophilic enzymes at low and moderate temperatures offers potential economic benefits. As of now lipases from Pseudozyma antarctica have been extensively studied to understand their unique thermal stability at 90°C and also because of its use in the pharmaceutical, agriculture, food, cosmetics and chemical industry. A few of the other enzymes which have been studied include extracellular alpha-amylase and glucoamylase from the yeast Pseudozyma antarctica (Candida antarctica), an extra-cellular protease from Cryptococcus humicola, an aspartyl proteinase from Cryptococcus humicola, a novel extracellular subtilase from Leucosporidium antarcticum, and a xylanase from Cryptococcus adeliensis

Expression of transforming growth factor alpha and epidermal growth factor receptor messenger RNA in neoplastic and nonneoplastic human kidney tissue.

PubMed

Mydlo, J H; Michaeli, J; Cordon-Cardo, C; Goldenberg, A S; Heston, W D; Fair, W R

1989-06-15

Using Northern blot analysis, we have demonstrated that mRNA for transforming growth factor alpha (TGF-alpha) was expressed in five malignant kidney tissue specimens but was not detected in their autologous nonneoplastic homologues. In addition, the expression of epidermal growth factor (EGF) receptor mRNA in these malignant tissues was 2- to 3-fold greater than in nontransformed tissues. In two cases examined using immunohistochemistry, we were able to correlate the increased expression of the mRNA with an increase in protein expression. Since TGF-alpha is known to bind to the EGF receptor, the finding of an increased expression of both TGF-alpha and EGF receptor mRNA in kidney tumor tissue suggests that interaction between TGF-alpha and the EGF receptor may play a role in promoting transformation and/or proliferation of kidney neoplasms, perhaps by an autocrine mechanism.

Specialist nectar-yeasts decline with urbanization in Berlin

NASA Astrophysics Data System (ADS)

Wehner, Jeannine; Mittelbach, Moritz; Rillig, Matthias C.; Verbruggen, Erik

2017-03-01

Nectar yeasts are common inhabitants of insect-pollinated flowers but factors determining their distribution are not well understood. We studied the influence of host identity, environmental factors related to pollution/urbanization, and the distance to a target beehive on local distribution of nectar yeasts within Robinia pseudoacacia L. and Tilia tomentosa Moench in Berlin, Germany. Nectar samples of six individuals per species were collected at seven sites in a 2 km radius from each target beehive and plated on YM-Agar to visualise the different morphotypes, which were then identified by sequencing a section of the 26S rDNA gene. Multivariate linear models were used to analyze the effects of all investigated factors on yeast occurrence per tree. Yeast distribution was mainly driven by host identity. The influence of the environmental factors (NO2, height of construction, soil sealing) strongly depended on the radius around the tree, similar to the distance of the sampled beehive. Incidence of specialist nectar-borne yeast species decreased with increasing pollution/urbanization index. Given that specialist yeast species gave way to generalist yeasts that have a reduced dependency on pollinators for between-flower dispersal, our results indicate that increased urbanization may restrict the movement of nectar-specialized yeasts, via limitations of pollinator foraging behavior.

Downdating a time-varying square root information filter

NASA Technical Reports Server (NTRS)

Muellerschoen, Ronald J.

1990-01-01

A new method to efficiently downdate an estimate and covariance generated by a discrete time Square Root Information Filter (SRIF) is presented. The method combines the QR factor downdating algorithm of Gill and the decentralized SRIF algorithm of Bierman. Efficient removal of either measurements or a priori information is possible without loss of numerical integrity. Moreover, the method includes features for detecting potential numerical degradation. Performance on a 300 parameter system with 5800 data points shows that the method can be used in real time and hence is a promising tool for interactive data analysis. Additionally, updating a time-varying SRIF filter with either additional measurements or a priori information proceeds analogously.

Enhanced actions of insulin-like growth factor-I and interferon-alpha co-administration in experimental cirrhosis.

PubMed

Tutau, Federico; Rodríguez-Ortigosa, Carlos; Puche, Juan Enrique; Juanarena, Nerea; Monreal, Iñigo; García Fernández, María; Clavijo, Encarna; Castilla, Alberto; Castilla-Cortázar, Inma

2009-01-01

Cirrhosis is a diffuse process of hepatic fibrosis and regenerative nodule formation. The liver is the major source of circulating insulin-like growth factor-I (IGF-I) whose plasma levels are diminished in cirrhosis. IGF-I supplementation has been shown to induce beneficial effects in cirrhosis, including antifibrogenic and hepatoprotective effects. On other hand, interferon-alpha (IFN-alpha) therapy seems to suppress the progression of hepatic fibrosis. The aim of this study was to investigate the effect of the co-administration of IGF-I+IFN-alpha to Wistar rats with CCl(4)-induced cirrhosis, exploring liver function tests, hepatic lipid peroxidation and histopathology. The mechanisms underlying the effects of these agents were studied by reverse transcription-polymerase chain reaction, determining the expression of some factors [hepatocyte growth factor (HGF), transforming growth factor-beta (TGF-beta), alpha-smooth muscle actin, collagen, tissular inhibitor of metalloproteinases-1 and pregnane X receptor (PXR)] involved in fibrogenesis, fibrolysis and/or hepatoprotection. Both IGF-I and IFN-alpha exerted significant effects on fibrogenesis. IGF-I significantly increased serum albumin and HGF whereas IFN-alpha-therapy did not. The inhibition of TGF-beta expression was only observed by the effect of IFN-alpha-therapy. In addition, only the co-administration of IGF-I and IFN-alpha was able to increase the PXR. The combined therapy with both factors improved liver function tests, hepatic lipid peroxidation and reduced fibrosis, inducing a relevant histological improvement, reducing fibrosis and recovering hepatic architecture. The co-administration IGF-I+IFN enhanced all the beneficial effects observed with each factor separately, showing an additive action on histopathology and PXR expression, which is involved in the inhibition of fibrogenesis.

Coordinate Regulation of Yeast Sterol Regulatory Element-binding Protein (SREBP) and Mga2 Transcription Factors.

PubMed

Burr, Risa; Stewart, Emerson V; Espenshade, Peter J

2017-03-31

The Mga2 and Sre1 transcription factors regulate oxygen-responsive lipid homeostasis in the fission yeast Schizosaccharomyces pombe in a manner analogous to the mammalian sterol regulatory element-binding protein (SREBP)-1 and SREBP-2 transcription factors. Mga2 and SREBP-1 regulate triacylglycerol and glycerophospholipid synthesis, whereas Sre1 and SREBP-2 regulate sterol synthesis. In mammals, a shared activation mechanism allows for coordinate regulation of SREBP-1 and SREBP-2. In contrast, distinct pathways activate fission yeast Mga2 and Sre1. Therefore, it is unclear whether and how these two related pathways are coordinated to maintain lipid balance in fission yeast. Previously, we showed that Sre1 cleavage is defective in the absence of mga2 Here, we report that this defect is due to deficient unsaturated fatty acid synthesis, resulting in aberrant membrane transport. This defect is recapitulated by treatment with the fatty acid synthase inhibitor cerulenin and is rescued by addition of exogenous unsaturated fatty acids. Furthermore, sterol synthesis inhibition blocks Mga2 pathway activation. Together, these data demonstrate that Sre1 and Mga2 are each regulated by the lipid product of the other transcription factor pathway, providing a source of coordination for these two branches of lipid synthesis. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Purification and characterization of an N alpha-acetyltransferase from Saccharomyces cerevisiae.

PubMed

Lee, F J; Lin, L W; Smith, J A

1988-10-15

N alpha-Acetyltransferase, which catalyzes the transfer of an acetyl group from acetyl coenzyme A to the alpha-NH2 group of proteins and peptides, was isolated from Saccharomyces cerevisiae and demonstrated by protein sequence analysis to be NH2-terminally blocked. The enzyme was purified 4,600-fold to apparent homogeneity by successive purification steps using DEAE-Sepharose, hydroxylapatite, DE52 cellulose, and Affi-Gel blue. The Mr of the native enzyme was estimated to be 180,000 +/- 10,000 by gel filtration chromatography, and the Mr of each subunit was estimated to be 95,000 +/- 2,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme has a pH optimum near 9.0, and its pI is 4.3 as determined by chromatofocusing on Mono-P. The enzyme catalyzed the transfer of an acetyl group to various synthetic peptides, including human adrenocorticotropic hormone (ACTH) (1-24) and its [Phe2] analogue, yeast alcohol dehydrogenase I (1-24), yeast alcohol dehydrogenase II (1-24), and human superoxide dismutase (1-24). These peptides contain either Ser or Ala as NH2-terminal residues which together with Met are the most commonly acetylated NH2-terminal residues (Persson, B., Flinta, C., von Heijne, G., and Jornvall, H. (1985) Eur. J. Biochem. 152, 523-527). Yeast enolase, containing a free NH2-terminal Ala residue, is known not to be N alpha-acetylated in vivo (Chin, C. C. Q., Brewer, J. M., and Wold, F. (1981) J. Biol. Chem. 256, 1377-1384), and enolase (1-24), a synthetic peptide mimicking the protein's NH2 terminus, was not acetylated in vitro by yeast acetyltransferase. The enzyme did not catalyze the N alpha-acetylation of other synthetic peptides including ACTH(11-24), ACTH(7-38), ACTH(18-39), human beta-endorphin, yeast superoxide dismutase (1-24). Each of these peptides has an NH2-terminal residue which is rarely acetylated in proteins (Lys, Phe, Arg, Tyr, Val, respectively). Among a series of divalent cations, Cu2+ and Zn2+ were demonstrated to be

Enantiomeric Excesses of Acid Labile Amino Acid Precursors of the Murchison Meteorite

NASA Astrophysics Data System (ADS)

Pizzarello, Sandra

1998-10-01

Amino acids present in carbonaceous chondrite are extracted in water in part as free compounds and in approximately equal part as acid labile precursors. On the assumption that they would be free of contamination, the precursors of two Murchison amino acids that have terrestrial occurrence, alanine and glutamic acid, have been targeted for analysis of their enantiomeric ratios. Pyroglutamic acid, the precursor of glutamic acid, was found with an L-enantiomeric excess comparable to that of the free acid, while alanine's precursor, N-acetyl alanine, appears approximately racemic. Also alpha-imino propioacetic acid, a proposed end product of alanine synthesis in the meteorite, was analyzed and found racemic.

Enantiomeric Excesses of Acid Labile Amino Acid Precursors of the Murchison Meteorite

NASA Technical Reports Server (NTRS)

Pizzarello, Sandra

1998-01-01

Amino acids present in carbonaceous chondrite are extracted in water in part as free compounds and in approximately equal part as acid labile precursors. On the assumption that they would be free of contamination, the precursors of two Murchison amino acids that have terrestrial occurrence, alanine and glutamic acid, have been targeted for analysis of their enantiomeric ratios. Pyroglutamic acid, the precursor of glutamic acid, was found with an L-enantiomeric excess comparable to that of the free acid, while alanine's precursor, N-acetyl alanine, appears approximately racemic. Also alpha-imino propioacetic acid, a proposed end product of alanine synthesis in the meteorite, was analyzed and found racemic.

Mitochondria mediate tumor necrosis factor-alpha/NF-kappaB signaling in skeletal muscle myotubes

NASA Technical Reports Server (NTRS)

Li, Y. P.; Atkins, C. M.; Sweatt, J. D.; Reid, M. B.; Hamilton, S. L. (Principal Investigator)

1999-01-01

Tumor necrosis factor-alpha (TNF-alpha) is implicated in muscle atrophy and weakness associated with a variety of chronic diseases. Recently, we reported that TNF-alpha directly induces muscle protein degradation in differentiated skeletal muscle myotubes, where it rapidly activates nuclear factor kappaB (NF-kappaB). We also have found that protein loss induced by TNF-alpha is NF-kappaB dependent. In the present study, we analyzed the signaling pathway by which TNF-alpha activates NF-kappaB in myotubes differentiated from C2C12 and rat primary myoblasts. We found that activation of NF-kappaB by TNF-alpha was blocked by rotenone or amytal, inhibitors of complex I of the mitochondrial respiratory chain. On the other hand, antimycin A, an inhibitor of complex III, enhanced TNF-alpha activation of NK-kappaB. These results suggest a key role of mitochondria-derived reactive oxygen species (ROS) in mediating NF-kappaB activation in muscle. In addition, we found that TNF-alpha stimulated protein kinase C (PKC) activity. However, other signal transduction mediators including ceramide, Ca2+, phospholipase A2 (PLA2), and nitric oxide (NO) do not appear to be involved in the activation of NF-kappaB.

Solar H-alpha features with hot onsets. III. Long fibrils in Lyman-alpha and with ALMA

NASA Astrophysics Data System (ADS)

Rutten, R. J.

2017-02-01

In H-alpha most of the solar surface is covered by dense canopies of long opaque fibrils, but predictions for quiet-Sun observations with ALMA have ignored this fact. Comparison with Ly-alpha suggests that the extraordinary opacity of H-alpha fibrils is caused by hot precursor events. Application of a recipe that assumes momentary Saha-Boltzmann extinction during their hot onset to millimeter wavelengths suggests that ALMA will observe H-alpha-like fibril canopies, not acoustic shocks underneath, and will yield data more interesting than if these canopies were transparent. An additional file is available at the end of the PDF file of this article.This study is offered as compliment to M.W.M. de Graauw. Our ways, objects, instruments and spectral domains parted after the 1970 eclipse but converge here.

Tumor necrosis factor-alpha inhibits stem cell factor-induced proliferation of human bone marrow progenitor cells in vitro. Role of p55 and p75 tumor necrosis factor receptors.

PubMed Central

Rusten, L S; Smeland, E B; Jacobsen, F W; Lien, E; Lesslauer, W; Loetscher, H; Dubois, C M; Jacobsen, S E

1994-01-01

Stem cell factor (SCF), a key regulator of hematopoiesis, potently synergizes with a number of hematopoietic growth factors. However, little is known about growth factors capable of inhibiting the actions of SCF. TNF-alpha has been shown to act as a bidirectional regulator of myeloid cell proliferation and differentiation. This study was designed to examine interactions between TNF-alpha and SCF. Here, we demonstrate that TNF-alpha potently and directly inhibits SCF-stimulated proliferation of CD34+ hematopoietic progenitor cells. Furthermore, TNF-alpha blocked all colony formation stimulated by SCF in combination with granulocyte colony-stimulating factor (CSF) or CSF-1. The synergistic effect of SCF observed in combination with GM-CSF or IL-3 was also inhibited by TNF-alpha, resulting in colony numbers similar to those obtained in the absence of SCF. These effects of TNF-alpha were mediated through the p55 TNF receptor, whereas little or no inhibition was signaled through the p75 TNF receptor. Finally, TNF-alpha downregulated c-kit cell-surface expression on CD34+ bone marrow cells, and this was predominantly a p55 TNF receptor-mediated event as well. Images PMID:7518828

Evidence for a single class of somatostatin receptors in ground squirrel cerebral cortex

DOE Office of Scientific and Technical Information (OSTI.GOV)

Krantic, S.; Petrovic, V.M.; Quirion, R.

1989-01-01

In the present study we characterized high-affinity somatostatin (SRIF) binding sites (Kd = 2.06 +/- 0.32 nM and Bmax = 295 +/- 28 fmol/mg protein) in cerebral cortex membrane preparations of European ground squirrel using /sup 125/I-(Tyr0-D-Trp8)-SRIF14 as a radioligand. The inhibition of radioligand specific binding by SRIF14, as well as by its agonists (SRIF28, Tyr0-D-Trp8-SRIF14, SMS 201 995) was complete and monophasic, thus revealing a single population of somatostatinergic binding sites. Radioautographic analysis of /sup 125/I-(Tyr0-D-Trp8)-SRIF14 labeled brain sections confirmed the results of our biochemical study. The homogeneity of SRIF binding sites in the ground squirrel neocortex was notmore » dependent on the animal's life-cycle phase.« less

Characterization of the primary interaction between the mating pheromone, alpha-factor, and its receptor in Saccharomyces cerevisiae

DOE Office of Scientific and Technical Information (OSTI.GOV)

Raths, S.K.

1987-01-01

Alpha-factor is a peptide of thirteen amino acids which is required for mating between the haploid mating types, a and ..cap alpha.., in Saccharomyces cerevisiae. An analogue of alpha-factor, DHP/sup 8/ DHP/sup 11/ Nle/sup 12/ tridecapeptide, was catalytically reduced in the presence of /sup 3/H gas for production of a radiolabeled pheromone suitable for use in binding studies. Incorporation of tritium resulted in /sup 3/H-alpha-factor with high specific activity, purity, biological activity and long shelf-life. Binding studies revealed that alpha-factor interacts with its receptor via a simple, reversible process which obeys the law of mass action. Association and dissociation kineticsmore » indicate values of 2.92 x 10/sup 6/ M/sup /minus/1/ min/sup -1/ for k/sub 1/ and between 4 and 7 x 10/sup /minus/2/ min/sup /minus/1/ for k/sub /minus/1/. Saturation binding studies reveal an equilibrium dissociation constant equal to 2.32 x 10/sup /minus/8/ M which approximate the kinetically-derived K/sub D/ of 2.12 x 10/sup /minus/8/ M. Scatchard and Hill analyses as well as dissociation behavior in the presence of excess unlabeled ligand indicate alpha-factor interacts with a homogeneous population of binding sites which do not interact and exhibit one affinity for the alpha-factor pheromone.« less

Characterization of synthesis and storage of TGF-alpha in rat parotid acinar and intercalated duct cells.

PubMed

Login, G R; Yang, J; Bryan, K P; Digenis, E C; McBride, J; Elovic, A; Quissell, D O; Dvorak, A M; Wong, D T

1997-03-01

Although the expression and biological role of transforming growth factor-alpha (TGF-alpha) have been explored in a variety of normal cells in mammalian species, little is known about the storage of TGF-alpha in secretory cells of exocrine organs. Parotid glands from four rats were homogenized for RNA isolation followed by reverse transcription-polymerase chain reaction to determine the presence of TGF-alpha message. In situ hybridization using a hamster-specific TGF-alpha riboprobe was done on paraffin sections. Parotid gland and isolated acinar cells were processed for transmission electron microscopy (TEM) and postembedding immunogold labeled for TGF-alpha. Gold particles were counted on approximately 200 granules in 10 acinar cells and in 10 intercalated duct cells. Labeling density was calculated as the number of gold particles per square micrometer +/- SD. Statistical significance was calculated using one-way analysis of variance. Using multiple technologies, we have established that rat parotid acinar and intercalated duct cells synthesize TGF-alpha and store the precursor form of this cytokine in their secretory granules.

Application of genetics to the development of starch-fermenting yeasts

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mattoon, J.R.; Kim, K.; Laluce, C.

1987-01-01

Yeast strains capable of direct fermentation of manioc starch were developed by hybridizing strains of Saccharomyces diastaticus and Saccharomyces cerevisiae. Hybrids were evaluated for speed of alcohol production, and yields and speed of formation of glycoamylase. Up to 6% solutions of Lintner starch could be fermented directly with about 80% conversion to alcohol. Pretreatment of crude 40% manioc starch suspensions with alpha-amylase, followed by fermentations with a starch-fermenting yeast strain, permitted accumulation of 12% ethanol within three days. Starch conversion was almost 100%. A fragment of DNA was cloned from S. diastaticus using the yeast-E. coli shuttle vector, YEp13, andmore » was used to transform a strain of S. cerevisiae to a starch-fermenting state. Supported by National Science Foundation grant INT 7927328 and National Institutes of Health grant GM 27860. Dr. Laluce was supported by a grant from Fundacao de Amparo a Pesquisa do Estado do Sao Paulo and by her university. (Refs. 5).« less

Nrt1 and Tna1-Independent Export of NAD+ Precursor Vitamins Promotes NAD+ Homeostasis and Allows Engineering of Vitamin Production

PubMed Central

Belenky, Peter; Stebbins, Rebecca; Bogan, Katrina L.; Evans, Charles R.; Brenner, Charles

2011-01-01

NAD+ is both a co-enzyme for hydride transfer enzymes and a substrate of sirtuins and other NAD+ consuming enzymes. NAD+ biosynthesis is required for two different regimens that extend lifespan in yeast. NAD+ is synthesized from tryptophan and the three vitamin precursors of NAD+: nicotinic acid, nicotinamide and nicotinamide riboside. Supplementation of yeast cells with NAD+ precursors increases intracellular NAD+ levels and extends replicative lifespan. Here we show that both nicotinamide riboside and nicotinic acid are not only vitamins but are also exported metabolites. We found that the deletion of the nicotinamide riboside transporter, Nrt1, leads to increased export of nicotinamide riboside. This discovery was exploited to engineer a strain to produce high levels of extracellular nicotinamide riboside, which was recovered in purified form. We further demonstrate that extracellular nicotinamide is readily converted to extracellular nicotinic acid in a manner that requires intracellular nicotinamidase activity. Like nicotinamide riboside, export of nicotinic acid is elevated by the deletion of the nicotinic acid transporter, Tna1. The data indicate that NAD+ metabolism has a critical extracellular element in the yeast system and suggest that cells regulate intracellular NAD+ metabolism by balancing import and export of NAD+ precursor vitamins. PMID:21589930

Renal alpha-smooth muscle actin: a new prognostic factor for lupus nephritis.

PubMed

Makni, Kaouthar; Jarraya, Faïçal; Khabir, Abdelmajid; Hentati, Basma; Hmida, Mohamed Ben; Makni, Hafedh; Boudawara, Tahia; Jlidi, Rchid; Hachicha, Jamil; Ayadi, Hammadi

2009-08-01

Systemic lupus erythematosus (SLE) is the prototype of autoimmune disease where renal involvement is frequent and always severe. Histological prognostic factors proposed for lupus nephritis (LN) including the World Health Organization and International Society of Nephrology/Renal Pathology Society--Working Group on the Classification classifications, active (AI) and chronicity (CI) indices may not predict response to treatment. The aim of this study was to correlate alpha-smooth muscle actin (alpha-SMA) expression, an early marker of glomerular and interstitial response to injury, to AI and CI, renal scarring progression and response to treatment. Fifty-seven kidney biopsy specimens obtained from 32 patients suffering from LN were studied. Twenty patients with class IV LN at first biopsy were identified to study renal progression to chronic renal failure until the use of immunosuppressive treatment. Interstitial alpha-SMA (I-alpha-SMA) was correlated only with CI (P < 0.001) whereas mesangial alpha-SMA (M-alpha-SMA) was correlated with neither LN activity (P = 0.126) nor sclerosis (P = 0.297). Only I-alpha-SMA was correlated with renal failure (P = 0.01). We divided patients with class IV LN into progressors and non-progressors based on the slope of serum creatinine. At first biopsy, M-alpha-SMA and I-alpha-SMA, but not AI and CI, were correlated with renal failure progression (M-alpha-SMA, 9.7b1.1 vs 7.8b1.4, P = 0.004; and I-alpha-SMA, 9.3b1.1 vs 6.5b3.2, P = 0.011). The study data highlight that I-alpha-SMA immunostain in class IV LN patients was correlated with chronicity indices. Moreover, M-alpha-SMA and I-alpha-SMA expression in first biopsy predicted renal progression modality. alpha-SMA expression may therefore be a useful marker to predict renal prognosis in LN.

Circulating tumour necrosis factor alpha & soluble TNF receptors in patients with Guillain-Barre syndrome.

PubMed

Radhakrishnan, V V; Sumi, M G; Reuben, S; Mathai, A; Nair, M D

2003-05-01

Tumour necrosis factor-alpha (TNF-alpha) is regarded as one of the immune factors that can induce demyelination of peripheral nerves in patients with Guillian-Barre syndrome (GBS). This present study was undertaken to find out the role of TNF-alpha and soluble TNF receptors in the pathogenesis of GBS; and to study the effect of intravenous immunoglobulin (ivIg) therapy on the serum TNF-alpha and soluble TNF receptors in patients with GBS. Thirty six patients with GBS in progressive stages of motor weakness were included in this study. The serum TNF-alpha and soluble TNF receptors (TNF-RI, TNF-RII) were measured in the serum samples of these patients before and after ivIg therapy by a sandwich ELISA. Of the 36 patients with GBS, 26 (72.2%) showed elevated serum TNF-alpha levels prior to ivIg therapy. Following a complete course of ivIg therapy there was a progressive decrease in the serum TNF-alpha concentrations in these 26 patients. On the other hand, the soluble TNF receptors, particularly TNF-RII showed an increase in the serum of GBS patients following ivIg therapy. The results indicate that ivIg reduces the serum TNF-alpha concentrations in the GBS patients having elevated levels prior to ivIg therapy. Elevated serum levels of soluble TNF receptors following ivIg therapy may play a protective role by inhibiting the demyelinating effect of TNF-alpha in the peripheral nerves of patients with GBS.

Interleukin-10 to tumor necrosis factor-alpha ratio is a predictive biomarker in nonalcoholic fatty liver disease: interleukin-10 to tumor necrosis factor-alpha ratio in steatohepatitis.

PubMed

Hashem, Reem M; Mahmoud, Mona F; El-Moselhy, Mohamed A; Soliman, Hala M

2008-10-01

Fatty liver disease is commonly associated with diabetes mellitus (DM). Insulin resistance (IR) as an investigative biomarker is only concerned with fatty liver that results from DM type 2 associated with metabolic syndrome. Irrespective of IR, DM is generally characterized by overproduction of the proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha), whereas action of the latter is modulated by the anti-inflammatory cytokine interleukin-10 (IL-10). The aim of this study was to investigate the efficacy of using TNF-alpha alone or IL-10/TNF-alpha ratio compared to IR, as a promising biomarker for fatty liver assessment in DM. Furthermore, we hypothesized that using garlic as an immunomodulator may decrease TNF-alpha and increase IL-10 production to improve steatohepatitis. DM was induced metabolically by a high-fat diet to bring about IR, or chemically by alloxan, producing insulin deficiency, in male albino rats. Garlic powder was supplemented (15 mg/kg per day) for 3 weeks. Fatty liver was depicted histologically and biochemically (aspartic aminotransferase, alanine aminotransferase, HOMA-IR, TNF-alpha, IL-10, IL-10/TNF-alpha ratio). We found that, in contrast to obese rats, garlic decreased IL-10/TNF-alpha ratio, despite decreasing TNF-alpha in alloxan diabetic rats in agreement with the histology, which revealed more prominent improvement in the obese group. Moreover, the effect of garlic was not linked to improvement of IR in obese rats. We conclude that IL-10/TNF-alpha ratio may be considered as a convenient biomarker for investigation of fatty liver of different grades, apart from being associated with IR, and immunomodulation of this ratio in favor of increasing it may exert significant improvement.

Synthesis of a beta-estradiol-biotin chimera that potently heterodimerizes estrogen receptor and streptavidin proteins in a yeast three-hybrid system.

PubMed

Hussey, Stephen L; Muddana, Smita S; Peterson, Blake R

2003-04-02

Small molecules that dimerize proteins in living cells provide powerful probes of biological processes and have potential as tools for the identification of protein targets of natural products. We synthesized 7-alpha-substituted derivatives of beta-estradiol tethered to the natural product biotin to regulate heterodimerization of estrogen receptor (ER) and streptavidin (SA) proteins expressed as components of a yeast three-hybrid system. Addition of an estradiol-biotin chimera bearing a 19-atom linker to yeast expressing DNA-bound ER-alpha or ER-beta LexA fusion proteins and wild-type SA protein fused to the B42 activation domain activated reporter gene expression by as much as 450-fold in vivo (10 muM ligand). Comparative analysis of lower affinity Y43A (biotin Kd approximately 100 pM) and W120A (biotin Kd approximately 100 nM) mutants of SA indicated that moderate affinity interactions can be readily detected with this system. Comparison of a 7-alpha-substituted estradiol-biotin chimera with a structurally similar dexamethasone-biotin chimera revealed that yeast expressing ER proteins can detect cognate ligands with up to 5-fold greater potency and 70-fold higher activity than yeast expressing analogous glucocorticoid receptor (GR) proteins. This approach may facilitate the identification of protein targets of biologically active small molecules screened against genetically encoded libraries of proteins expressed in yeast three-hybrid systems.

[Alpha-melanocyte-stimulating hormone. From bench to bedside].

PubMed

Böhm, M; Luger, T A

2010-06-01

Alpha-melanocyte-stimulating hormone (alpha-MSH) is a tridecapeptide that is produced by the skin itself from the precursor proopiomelanocortin. It crucially mediates ultraviolet light-induced tanning after binding to melanocortin-1 receptors (MC-1R) expressed on the surface of epidermal melanocytes. The potent pigment-inducing and also cytoprotective actions of alpha-MSH are the rationale for the performance of first phase II clinical trials with Nle4-D-Phe7-alpha-MSH (NDP-alpha-MSH), a subcutaneously administered synthetic and superpotent alpha-MSH analogue, in patients with photodermatoses such as erythropoietic protoporphyria. Since alpha-MSH has shown promising anti-inflammatory and antifibrotic properties in numerous preclinical studies, it will be most interesting to evaluate these effects in further clinical pilot studies with NDP-alpha-MSH. In addition to alpha-MSH analogues, truncated tripeptides such as KDPT which do not bind to MC-1R but have sustained anti-inflammatory properties are currently emerging as another novel therapeutic strategy in dermatology.

Tumour necrosis factor-alpha polymorphism as one of the complex inherited factors in pemphigus.

PubMed Central

Torzecka, Jolanta Dorota; Narbutt, Joanna; Sysa-Jedrzejowska, Anna; Borowiec, Maciej; Ptasinska, Anetta; Woszczek, Grzegorz; Kowalski, Marek L

2003-01-01

The aim of our study was to analyse a significance of tumour necrosis factor (TNF)-alpha promoter gene polymorphisms in relation to the HLA-DR locus in genetic predisposition to pemphigus. TNF-alpha gene polymorphisms in position -238 and -308 were identified using a modified polymerase chain reaction-restriction fragment length polymorphism method in 53 patients with pemphigus (38 with pemphigus vulgaris, 15 with pemphigus foliaceus) and 87 healthy controls. The HLA-DRB1 locus was typed using the polymerase chain reaction SSO method in all the patients and 152 population controls. Carriers of the TNF-alpha polymorphic -308 A allele were found to be more frequent in the pemphigus foliaceus group in comparison with the control group (odds ratio (OR) = 8.12; p = 0.0005). A significant association between HLA-DRB1*04 (OR = 3.86; pcor = 0.0001) and DRB1*14 (OR = 8.4; pcor = 0.0001) and pemphigus vulgaris was found. In this group of patients a decreased frequency of HLA-DRB1*07 (OR = 0.08; pcor = 0.006) was also identified. We have shown for the first time a positive association of TNF-alpha polymorphism in position -308 with pemphigus foliaceus. PMID:14760938

Tumour necrosis factor-alpha polymorphism as one of the complex inherited factors in pemphigus.

PubMed

Torzecka, Jolanta Dorota; Narbutt, Joanna; Sysa-Jedrzejowska, Anna; Borowiec, Maciej; Ptasinska, Anetta; Woszczek, Grzegorz; Kowalski, Marek L

2003-10-01

The aim of our study was to analyse a significance of tumour necrosis factor (TNF)-alpha promoter gene polymorphisms in relation to the HLA-DR locus in genetic predisposition to pemphigus. TNF-alpha gene polymorphisms in position -238 and -308 were identified using a modified polymerase chain reaction-restriction fragment length polymorphism method in 53 patients with pemphigus (38 with pemphigus vulgaris, 15 with pemphigus foliaceus) and 87 healthy controls. The HLA-DRB1 locus was typed using the polymerase chain reaction SSO method in all the patients and 152 population controls. Carriers of the TNF-alpha polymorphic -308 A allele were found to be more frequent in the pemphigus foliaceus group in comparison with the control group (odds ratio (OR) = 8.12; p = 0.0005). A significant association between HLA-DRB1*04 (OR = 3.86; pcor = 0.0001) and DRB1*14 (OR = 8.4; pcor = 0.0001) and pemphigus vulgaris was found. In this group of patients a decreased frequency of HLA-DRB1*07 (OR = 0.08; pcor = 0.006) was also identified. We have shown for the first time a positive association of TNF-alpha polymorphism in position -308 with pemphigus foliaceus.

A Serendipitous Discover that in situ Proteolysis is Essential for the Crystallization of Yeast CPSF-100 (Ydh1p)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mandel,C.; Gebauer, D.; Zhang, H.

2006-01-01

The cleavage and polyadenylation specificity factor (CPSF) complex is required for the cleavage and polyadenylation of the 3'-end of messenger RNA precursors in eukaryotes. During structural studies of the 100 kDa subunit (CPSF-100, Ydh1p) of the yeast CPSF complex, it was serendipitously discovered that a solution that is infected by a fungus (subsequently identified as Penicillium) is crucial for the crystallization of this protein. Further analyses suggest that the protein has undergone partial proteolysis during crystallization, resulting in the deletion of an internal segment of about 200 highly charged and hydrophilic residues, very likely catalyzed by a protease secreted bymore » the fungus. With the removal of this segment, yeast CPSF-100 (Ydh1p) has greatly reduced solubility and can be crystallized in the presence of a minute amount of precipitant.« less

In vitro bioactivity of 17alpha-estradiol.

PubMed

Sievernich, André; Wildt, Ludwig; Lichtenberg-Fraté, Hella

2004-12-01

A miniaturised short-term in vitro assay based on the activation of the human estrogen receptor alpha and genetically modified yeast (Saccharomyces cerevisiae) cells was performed to explore the capacity of this system to monitor the bioactivity of estrogenic compounds, particularly 17alpha- and 17beta-estradiol. Together with the human estrogen receptor (hER)-alpha plasmid, the reporter plasmid containing a yeast-optimised version of the green fluorescent protein (yEGFP) linked to three repeats of the cis-acting estrogen hormone-responsive element (ERE) were expressed in a strain being deleted in the pleiotropic drug resistance transporters Pdr5, Snq2 and Yor1, known to facilitate efflux of organic compounds including steroids and chemotherapeutics. Agonists that bind to hER in vitro trigger estrogen receptor-mediated transcriptional activation of the GFP reporter gene monitored by fluorescence emission at 535 nm. The sensitivity of the assay was tested with various 17alpha- and 17beta-estradiol concentrations, yielding a detection limit of 5 pg/ml (0.018 nM) for the agonist 17beta-E2 in solvent and in human charcoal-stripped serum using a S. cerevisiae pdr5, snq2 and yor1 mutant strain. For 17alpha-estradiol only, at approximately 1500 pg/ml a similar fluorescence response compared to 100 pg/ml 17beta-E2 was observed implicating a much weaker potency of this stereoisomer. The specificity of the system was tested by expression of a truncated hER lacking the ligand-binding domain E and by administration of the androgen, 4-androsten 3,17 dione. Both controls did not yield an increase in fluorescence emission. This fluorescence emission assay enables detection of estrogenic biological activity induced by direct agonists, such as 17beta-E2 at concentrations similar to those found in human sera or by estrogen-like chemicals.

Alpha decay hindrance factors and reflection asymmetry in nuclei

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sheline, R.K.; Bossinga, B.B.

1991-07-01

All available hindrance factors of alpha transitions to low-lying negative-parity states in doubly even nuclei, to odd-{ital A} parity doublets and to doubly odd parity doublet bands, are used to study the systematics of reflection asymmetry in the {ital A}{similar to}218--230 region. Special attention is given to the polarization effect of the odd particle in increasing reflection asymmetry and therefore decreasing hindrance factors to the opposite parity states of octupole bands.

Human gut Bacteroidetes can utilize yeast mannan through a selfish mechanism

USDA-ARS?s Scientific Manuscript database

The architecture of the human distal gut microbiota (microbiota) is sculpted by the complex carbohydrates delivered in the diet. Yeasts, which are among the earliest domesticated microorganisms and have been a component of the human diet for at least 7000 years, possess an elaborate cell wall alpha-...

Pathogen effector protein screening in yeast identifies Legionella factors that interfere with membrane trafficking.

PubMed

Shohdy, Nadim; Efe, Jem A; Emr, Scott D; Shuman, Howard A

2005-03-29

Legionella pneumophila invades and replicates intracellularly in human and protozoan hosts. The bacteria use the Icm/Dot type IVB secretion system to translocate effectors that inhibit phagosome maturation and modulate host vesicle trafficking pathways. To understand how L. pneumophila modulates organelle trafficking in host cells, we carried out pathogen effector protein screening in yeast, identifying L. pneumophila genes that produced membrane trafficking [vacuole protein sorting (VPS)] defects in yeast. We identified four L. pneumophila DNA fragments that perturb sorting of vacuolar proteins. Three encode ORFs of unknown function that are translocated via the Icm/Dot transporter from Legionella into macrophages. VPS inhibitor protein (Vip) A is a coiled-coil protein, VipD is a patatin domain-containing protein, and VipF contains an acetyltransferase domain. Processing studies in yeast indicate that VipA, VipD, and VipF inhibit lysosomal protein trafficking by different mechanisms; overexpressing VipA has an effect on carboxypeptidase Y trafficking, whereas VipD interferes with multivesicular body formation at the late endosome and endoplasmic reticulum-to-Golgi body transport. Such differences highlight the multiple strategies L. pneumophila effectors use to subvert host trafficking processes. Using yeast as an effector gene discovery tool allows for a powerful, genetic approach to both the identification of virulence factors and the study of their function.

The effect of pasteurization on transforming growth factor alpha and transforming growth factor beta 2 concentrations in human milk.

PubMed

McPherson, R J; Wagner, C L

2001-01-01

Transforming growth factor alpha (TGF-alpha) and beta 2 (TGF-beta2) are present in human milk and are involved in growth differentiation and repair of neonatal intestinal epithelia. Heat treatment at 56 degrees C has been shown effective for providing safe banked donor milk, with good retention of other biologically active factors. The purpose of our study was to determine the effect of heat sterilization on TGF-alpha and TGF-beta2 concentrations in human milk. Twenty milk samples were collected from 20 lactating mothers in polypropylene containers and frozen at -20 degrees C for transport or storage. Before heat treatment by holder pasteurization, the frozen milk was thawed and divided into 1-mL aliquots. All samples were heated in an accurately regulated water bath until a holding temperature was achieved, then held for 30 minutes using constant agitation. Holding temperature ranged from 56.5 degrees C to 56.9 degrees C. The milk was then stored at 4 degrees C overnight for analysis the following day. The concentration of TGF-alpha was measured by radioimmunoassay. Mean concentration +/- SD of TGF-alpha in raw milk samples was 119+/-50 pg/mL, range 57 to 234. The mean concentration +/- SD of TGF-alpha in heat treated samples was 113+/-50 pg/mL, range 51 to 227. TGF-alpha concentration was minimally affected by pasteurization, with an overall loss of 6.1%. Of 19 samples, 4 had increased and 15 had decreased concentrations after pasteurization (mean percent SEM: 94%+/-7% of raw milk, range 72%+/-107%). The concentration of acid-activated TGF-beta2 was measured by enzyme-linked immunosorbent assay. Mean concentration +/- SD of TGF-beta2 in raw milk samples was 5624+/-5038 pg/mL, range 195 to 15480. The mean concentration +/- SD of TGF-beta2 in heat-treated samples was 5073+/-4646 pg/mL, range 181 to 15140. TGF-beta2 survived with relatively little loss (0.6%): of 18 samples, 11 had increased and 7 had decreased concentrations after pasteurization (mean percent

Retrograde Signaling as a Mechanism of Yeast Adaptation to Unfavorable Factors.

PubMed

Trendeleva, T A; Zvyagilskaya, R A

2018-02-01

Mitochondria perform many essential functions in eukaryotic cells. Being the main producers of ATP and the site of many catabolic and anabolic reactions, they participate in intracellular signaling, proliferation, aging, and formation of reactive oxygen species. Mitochondrial dysfunction is the cause of many diseases and even cell death. The functioning of mitochondria in vivo is impossible without interaction with other cellular compartments. Mitochondrial retrograde signaling is a signaling pathway connecting mitochondria and the nucleus. The major signal transducers in the yeast retrograde response are Rtg1p, Rtg2p, and Rtg3p proteins, as well as four additional negative regulatory factors - Mks1p, Lst8p, and two 14-3-3 proteins (Bmh1/2p). In this review, we analyze current information on the retrograde signaling in yeast that is regarded as a stress or homeostatic response mechanism to changes in various metabolic and biosynthetic activities that occur upon mitochondrial dysfunction. We also discuss relations between retrograde signaling and other signaling pathways in the cell.

PCNA mono-ubiquitination and activation of translesion DNA polymerases by DNA polymerase {alpha}.

PubMed

Suzuki, Motoshi; Niimi, Atsuko; Limsirichaikul, Siripan; Tomida, Shuta; Miao Huang, Qin; Izuta, Shunji; Usukura, Jiro; Itoh, Yasutomo; Hishida, Takashi; Akashi, Tomohiro; Nakagawa, Yoshiyuki; Kikuchi, Akihiko; Pavlov, Youri; Murate, Takashi; Takahashi, Takashi

2009-07-01

Translesion DNA synthesis (TLS) involves PCNA mono-ubiquitination and TLS DNA polymerases (pols). Recent evidence has shown that the mono-ubiquitination is induced not only by DNA damage but also by other factors that induce stalling of the DNA replication fork. We studied the effect of spontaneous DNA replication errors on PCNA mono-ubiquitination and TLS induction. In the pol1L868F strain, which expressed an error-prone pol alpha, PCNA was spontaneously mono-ubiquitinated. Pol alpha L868F had a rate-limiting step at the extension from mismatched primer termini. Electron microscopic observation showed the accumulation of a single-stranded region at the DNA replication fork in yeast cells. For pol alpha errors, pol zeta participated in a generation of +1 frameshifts. Furthermore, in the pol1L868F strain, UV-induced mutations were lower than in the wild-type and a pol delta mutant strain (pol3-5DV), and deletion of the RAD30 gene (pol eta) suppressed this defect. These data suggest that nucleotide misincorporation by pol alpha induces exposure of single-stranded DNA, PCNA mono-ubiquitination and activates TLS pols.

A review of the alpha-1 foundation: its formation, impact, and critical success factors.

PubMed

Walsh, John W; Snider, Gordon L; Stoller, James K

2006-05-01

Patient-advocacy organizations have proliferated because they can be an effective method to advance research and clinical care for those with the index condition, and can produce substantial benefits for the affected community, especially when the condition is uncommon. To clarify critical success factors in organizing a patient-advocacy organization and to provide a blueprint for others, including the respiratory-care advocacy community, this report examines features of one highly successful organization, the Alpha-1 Foundation, which is committed to helping those with the genetic condition alpha-1 antitrypsin deficiency. Features of the Alpha-1 Foundation that underlie its success include: consistently creating partnerships with key stakeholders, including the scientific and clinical communities, government, and pharmaceutical manufacturers; bringing passion to the cause (eg, by assuring that organizational leadership is provided by individuals affected by alpha-1 antitrypsin deficiency); and developing strategic business partnerships, as with a company that administers alpha-1 antitrypsin treatment (so-called intravenous augmentation therapy) and employs individuals with alpha-1 antitrypsin deficiency. Funds allocated by the company help to underwrite the foundation's research-funding commitment. The foundation also recruits and retains talent, including alpha-1 patients, to leadership roles (eg, on the board of directors) and has a voluntary group of committed scientists and clinicians. We believe that attention to these factors can help assure the success of patient-advocacy groups.

Yeast as a model system for mammalian seven-transmembrane segment receptors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jeansonne, N.E.

1994-05-01

Investigators have used the budding yeast Saccharomyces cerevisiae as a model system in which to study the {beta}-adrenergic receptor, the T-cell receptor pathway, initiation of mammalian DNA replication, initiation of mammalian transcription, secretion, the CDC2 kinase system, cell cycle control, and aging, as well as the function of oncogenes. This list continues to growth with the discovery of an immunoglobulin heavy-chain binding homologue in yeast, an Rb binding protein homologue, and a possible yeast arrestin. Yeast is relatively easy to maintain, to grow, and to genetically manipulate. A single gene can be overexpressed, selectively mutated or deleted from its chromosomalmore » location. In this way, the in vivo function of a gene can be studied. It has become reasonable to consider yeast as a model system for studying the seven transmembrane segments (7-TMS) receptor family. Currently, subtypes of the {beta}-adrenergic receptor are being studied in yeast. The receptor and its G{sub {alpha}}-G-protein, trigger the mating pheromone receptor pathway. This provides a powerful assay for determining receptor function. Studies expressing the muscarinic cholinergic receptor in yeast are underway. The yeast pheromone receptor belongs to this receptor family, sharing sequences and secondary structure homology. An effective strategy has been to identify a yeast pathway or process which is homologous to a mammalian system. The pathway is delineated in yeast, identifying other genetic components. Then yeast genes are used to screen for human homologues of these components. The putative human homologues are then expressed in yeast and in mammalian cells to determine function. When this type of {open_quotes}mixing and matching{close_quotes} works, yeast genetics can be a powerful tool. 115 refs.« less

Comparison of melibiose utilizing baker's yeast strains produced by genetic engineering and classical breeding.

PubMed

Vincent, S F; Bell, P J; Bissinger, P; Nevalainen, K M

1999-02-01

Yeast strains currently used in the baking industry cannot fully utilize the trisaccharide raffinose found in beet molasses due to the absence of melibiase (alpha-galactosidase) activity. To overcome this deficiency, the MEL1 gene encoding melibiase enzyme was introduced into baker's yeast by both classical breeding and recombinant DNA technology. Both types of yeast strains were capable of vigorous fermentation in the presence of high levels of sucrose, making them suitable for the rapidly developing Asian markets where high levels of sugar are used in bread manufacture. Melibiase expression appeared to be dosage-dependent, with relatively low expression sufficient for complete melibiose utilization in a model fermentation system.

Ethyl Carbamate Formation Regulated by Lactic Acid Bacteria and Nonconventional Yeasts in Solid-State Fermentation of Chinese Moutai-Flavor Liquor.

PubMed

Du, Hai; Song, Zhewei; Xu, Yan

2018-01-10

This study aimed to identify specific microorganisms related to the formation of precursors of EC (ethyl carbamate) in the solid-state fermentation of Chinese Moutai-flavor liquor. The EC content was significantly correlated with the urea content during the fermentation process (R 2 = 0.772, P < 0.01). Differences in urea production and degradation were found at both species and functional gene levels by metatranscriptomic sequencing and culture-dependent analysis. Lactobacillus spp. could competitively degrade arginine through the arginine deiminase pathway with yeasts, and most Lactobacillus species were capable of degrading urea. Some dominant nonconventional yeasts, such as Pichia, Schizosaccharomyces, and Zygosaccharomyces species, were shown to produce low amounts of urea relative to Saccharomyces cerevisiae. Moreover, unusual urea degradation pathways (urea carboxylase, allophanate hydrolase, and ATP-independent urease) were identified. Our results indicate that EC precursor levels in the solid-state fermentation can be controlled using lactic acid bacteria and nonconventional yeasts.

Enhanced S-Adenosylmethionine Production by Increasing ATP Levels in Baker's Yeast ( Saccharomyces cerevisiae).

PubMed

Chen, Yawei; Tan, Tianwei

2018-05-23

In the biosynthesis of S-adenosylmethionine (SAM) in baker's yeast ( Saccharomyces cerevisiae), ATP functions as both a precursor and a driving force. However, few published reports have dealt with the control of ATP concentration using genetic design. In this study we have adopted a new ATP regulation strategy in yeast for enhancing SAM biosynthesis, including altering NADH availability and regulating the oxygen supply. Different ATP regulation systems were designed based on the introduction of water-forming NADH oxidase, Vitreoscilla hemoglobin, and phosphite dehydrogenase in combination with overexpression of the gene SAM2. Via application of this strategy, after 28 h cultivation, the SAM titer in the yeast strain ABYSM-2 reached a maximum level close to 55 mg/L, an increase of 67% compared to the control strain. The results show that the ATP regulation strategy is a valuable tool for SAM production and might further enhance the synthesis of other ATP-driven metabolites in yeast.

Tumor necrosis factor-alpha antagonists: differential clinical effects by different biotechnological molecules.

PubMed

Licastro, F; Chiappelli, M; Ianni, M; Porcellini, E

2009-01-01

Inhibitors of tumor necrosis factor-alpha have deeply changed the therapy of several inflammatory human diseases. For instance, clinical management of rheumatoid arthritis, psoriatic arthritis and ankylosing spondylitis have profoundly benefited after the introduction of new therapeutic tools, such as antagonist of TNF-alpha molecule. These drugs include etanercept, a soluble TNF-alpha receptor antagonist, three anti-TNF-alpha antibodies, adalimumab, infliximab, golimumab and certolizumab a humanized Fab fragment combined with polyethylene glycol. These compounds efficiently inhibit several TNF-alpha biological-mediated effects, however, they have also shown differential clinical efficacy in several trials from different autoimmune diseases. It is of clinical relevance that non-responders to one of these drugs often positively responded to another. Different mechanisms of action and diversity in pharmacokinetics of these three compounds may partially explain different clinical effects. However, partially diverse pathogenetic mechanisms in different diseases also contribute to differential therapeutic responses. Therefore, these apparently homogeneous agents can not be considered equivalent in their clinically efficacy. Differential therapeutic actions of these drugs may be advantageously used in clinical practice and further improve the great potential of individual TNF-alpha inhibitors.

Erythropoietin protects myocardin-expressing cardiac stem cells against cytotoxicity of tumor necrosis factor-{alpha}

DOE Office of Scientific and Technical Information (OSTI.GOV)

Madonna, Rosalinda; Institute of Cardiology, and Center of Excellence on Aging, 'G. d'Annunzio' University, Chieti; Shelat, Harnath

2009-10-15

Cardiac stem cells are vulnerable to inflammation caused by infarction or ischemic injury. The growth factor, erythropoietin (Epo), ameliorates the inflammatory response of the myocardium to ischemic injury. This study was designed to assess the role of Epo in regulation of expression and activation of the cell death-associated intracellular signaling components in cardiac myoblasts stimulated with the proinflammatory cytokine tumor necrosis factor (TNF)-{alpha}. Cardiac myoblasts isolated from canine embryonic hearts characterized by expression of myocardin A, a promyogenic transcription factor for cardiovascular muscle development were pretreated with Epo and then exposed to TNF-{alpha}. Compared to untreated cells, the Epo-treated cardiacmore » myoblasts exhibited better morphology and viability. Immunoblotting revealed lower levels of active caspase-3 and reductions in iNOS expression and NO production in Epo-treated cells. Furthermore, Epo pretreatment reduced nuclear translocation of NF-{kappa}B and inhibited phosphorylation of inhibitor of kappa B (I{kappa}B) in TNF-{alpha}-stimulated cardiac myoblasts. Thus, Epo protects cardiac myocyte progenitors or myoblasts against the cytotoxic effects of TNF-{alpha} by inhibiting NF-{kappa}B-mediated iNOS expression and NO production and by preventing caspase-3 activation.« less

Induction of autocrine factor inhibiting cell motility from murine B16-BL6 melanoma cells by alpha-melanocyte stimulating hormone.

PubMed

Murata, J; Ayukawa, K; Ogasawara, M; Watanabe, H; Saiki, I

1999-03-15

We have previously reported that neuropeptide alpha-melanocyte stimulating hormone (alpha-MSH) successfully inhibited Matrigel invasion and haptotactic migration of B16-BL6 melanoma cells towards both fibronectin and laminin without affecting their growth. In the present study, we investigated the inhibitory mechanism of tumor cell motility by alpha-MSH. Alpha-MSH significantly blocked the autocrine motility factor (AMF)-enhanced cell motility. However, alpha-MSH did neither prevent the secretion of AMF from B16-BL6 cells nor alter the expression level of AMF receptor (gp78). On the other hand, alpha-MSH induced the secretion of the motility inhibitory factor(s) from B16-BL6 cells in a concentration- and time-dependent manner. The induction of the motility inhibitor(s) was proportional to increasing levels of intracellular cAMP induced by alpha-MSH as well as forskolin, and the activity was abolished by an adenylate cyclase inhibitor, 2',5'-dideoxyadenosine (DDA). The motility-inhibiting activity in conditioned medium (CM) from alpha-MSH-treated B16-BL6 cells was found to have a m.w. below 3 kDa after fractionation. This activity was abolished by boiling but insensitive to trypsin. The treatment of tumor cells with cycloheximide reduced the activity in alpha-MSH-stimulated CM. Our results suggest that alpha-MSH inhibited the motility of B16-BL6 cells through induction of autocrine factor(s).

The rate of metabolism as a factor determining longevity of the Saccharomyces cerevisiae yeast.

PubMed

Molon, Mateusz; Szajwaj, Monika; Tchorzewski, Marek; Skoczowski, Andrzej; Niewiadomska, Ewa; Zadrag-Tecza, Renata

2016-02-01

Despite many controversies, the yeast Saccharomyces cerevisiae continues to be used as a model organism for the study of aging. Numerous theories and hypotheses have been created for several decades, yet basic mechanisms of aging have remained unclear. Therefore, the principal aim of this work is to propose a possible mechanism leading to increased longevity in yeast. In this paper, we suggest for the first time that there is a link between decreased metabolic activity, fertility and longevity expressed as time of life in yeast. Determination of reproductive potential and total lifespan with the use of fob1Δ and sfp1Δ mutants allows us to compare the "longevity" presented as the number of produced daughters with the longevity expressed as the time of life. The results of analyses presented in this paper suggest the need for a change in the definition of longevity of yeast by taking into consideration the time parameter. The mutants that have been described as "long-lived" in the literature, such as the fob1Δ mutant, have an increased reproductive potential but live no longer than their standard counterparts. On the other hand, the sfp1Δ mutant and the wild-type strain produce a similar number of daughter cells, but the former lives much longer. Our results demonstrate a correlation between the decreased efficiency of the translational apparatus and the longevity of the sfp1Δ mutant. We suggest that a possible factor regulating the lifespan is the rate of cell metabolism. To measure the basic metabolism of the yeast cells, we used the isothermal microcalorimetry method. In the case of sfp1Δ, the flow of energy, ATP concentration, polysome profile and translational fitness are significantly lower in comparison with the wild-type strain and the fob1Δ mutant.

Efficient production of hyperpolarized bicarbonate by chemical reaction on a DNP precursor to measure pH.

PubMed

Ghosh, Rajat K; Kadlecek, Stephen J; Pourfathi, Mehrdad; Rizi, Rahim R

2015-11-01

To produce hyperpolarized bicarbonate indirectly via chemical reaction from a hyperpolarized precursor and utilize it for the simultaneous regional measurement of metabolism and pH. Alpha keto carboxylic acids are first hyperpolarized by dissolution dynamic nuclear polarization (DNP). These precursor molecules are rapidly reacted with hydrogen peroxide (H2O2) to decarboxylate the species, resulting in new target molecules. Unreacted H2O2 is removed from the system by reaction with sulfite. Interrogation of the ratio of dissolved carbon dioxide (CO2) to bicarbonate can be used to determine pH. Conversion of hyperpolarized alpha keto acids to bicarbonate and CO2 results in a minimal loss of the spin order. The reaction can be conducted to completion within seconds and preserves the nuclear spin polarization. Through a rapid chemical reaction, we can conserve the nuclear spin order of a DNP precursor to generate multiple hyperpolarized bioprobes otherwise unamenable to polarization. This indirect technique for the production of hyperpolarized agents can be applied to different precursor compounds to generate additional novel probes. © 2014 Wiley Periodicals, Inc.

alpha1B-Adrenergic receptor phosphorylation and desensitization induced by transforming growth factor-beta.

PubMed Central

Romero-Avila, M Teresa; Flores-Jasso, C Fabián; García-Sáinz, J Adolfo

2002-01-01

Transforming growth factor-beta (TGF-beta) induced alpha(1B)-adrenergic receptor phosphorylation in Rat-1 fibroblasts stably expressing these adrenoceptors. This effect of TGF-beta was rapid, reaching a maximum within 30 min and decreasing thereafter, and concentration-dependent (EC(50) 0.3 pM). The phosphoinositide 3-kinase inhibitors wortmannin and LY294002, and the protein kinase C inhibitors staurosporine, Ro 318220 and bisindolylmaleimide, blocked the effect of this growth factor. alpha(1B)-Adrenergic receptor phosphorylation was associated with desensitization, as indicated by a reduction in the adrenergic-mediated production of [(3)H]inositol phosphates. Phosphorylation of alpha(1B)-adrenergic receptors by TGF-beta was also observed in Cos-1 cells transfected with the receptor. Co-transfection of the dominant-negative mutant of the regulatory subunit of phosphoinositide 3-kinase (Deltap85) inhibited the phosphorylation of alpha(1B)-adrenergic receptors induced by TGF-beta. Our results indicate that activation of TGF-beta receptors induces alpha(1B)-adrenergic receptor phosphorylation and desensitization. The data suggest that phosphoinositide 3-kinase and protein kinase C play key roles in this effect of TGF-beta. PMID:12234252

alpha1B-Adrenergic receptor phosphorylation and desensitization induced by transforming growth factor-beta.

PubMed

Romero-Avila, M Teresa; Flores-Jasso, C Fabián; García-Sáinz, J Adolfo

2002-12-01

Transforming growth factor-beta (TGF-beta) induced alpha(1B)-adrenergic receptor phosphorylation in Rat-1 fibroblasts stably expressing these adrenoceptors. This effect of TGF-beta was rapid, reaching a maximum within 30 min and decreasing thereafter, and concentration-dependent (EC(50) 0.3 pM). The phosphoinositide 3-kinase inhibitors wortmannin and LY294002, and the protein kinase C inhibitors staurosporine, Ro 318220 and bisindolylmaleimide, blocked the effect of this growth factor. alpha(1B)-Adrenergic receptor phosphorylation was associated with desensitization, as indicated by a reduction in the adrenergic-mediated production of [(3)H]inositol phosphates. Phosphorylation of alpha(1B)-adrenergic receptors by TGF-beta was also observed in Cos-1 cells transfected with the receptor. Co-transfection of the dominant-negative mutant of the regulatory subunit of phosphoinositide 3-kinase (Deltap85) inhibited the phosphorylation of alpha(1B)-adrenergic receptors induced by TGF-beta. Our results indicate that activation of TGF-beta receptors induces alpha(1B)-adrenergic receptor phosphorylation and desensitization. The data suggest that phosphoinositide 3-kinase and protein kinase C play key roles in this effect of TGF-beta.

Two-carbon metabolites, polyphenols and vitamins influence yeast chronological life span in winemaking conditions

PubMed Central

2012-01-01

Background Viability in a non dividing state is referred to as chronological life span (CLS). Most grape juice fermentation happens when Saccharomyces cerevisiae yeast cells have stopped dividing; therefore, CLS is an important factor toward winemaking success. Results We have studied both the physical and chemical determinants influencing yeast CLS. Low pH and heat shorten the maximum wine yeast life span, while hyperosmotic shock extends it. Ethanol plays an important negative role in aging under winemaking conditions, but additional metabolites produced by fermentative metabolism, such as acetaldehyde and acetate, have also a strong impact on longevity. Grape polyphenols quercetin and resveratrol have negative impacts on CLS under winemaking conditions, an unexpected behavior for these potential anti-oxidants. We observed that quercetin inhibits alcohol and aldehyde dehydrogenase activities, and that resveratrol performs a pro-oxidant role during grape juice fermentation. Vitamins nicotinic acid and nicotinamide are precursors of NAD+, and their addition reduces mean longevity during fermentation, suggesting a metabolic unbalance negative for CLS. Moreover, vitamin mix supplementation at the end of fermentation shortens CLS and enhances cell lysis, while amino acids increase life span. Conclusions Wine S. cerevisiae strains are able to sense changes in the environmental conditions and adapt their longevity to them. Yeast death is influenced by the conditions present at the end of wine fermentation, particularly by the concentration of two-carbon metabolites produced by the fermentative metabolism, such as ethanol, acetic acid and acetaldehyde, and also by the grape juice composition, particularly its vitamin content. PMID:22873488

Yeasts in sustainable bioethanol production: A review.

PubMed

Mohd Azhar, Siti Hajar; Abdulla, Rahmath; Jambo, Siti Azmah; Marbawi, Hartinie; Gansau, Jualang Azlan; Mohd Faik, Ainol Azifa; Rodrigues, Kenneth Francis

2017-07-01

Bioethanol has been identified as the mostly used biofuel worldwide since it significantly contributes to the reduction of crude oil consumption and environmental pollution. It can be produced from various types of feedstocks such as sucrose, starch, lignocellulosic and algal biomass through fermentation process by microorganisms. Compared to other types of microoganisms, yeasts especially Saccharomyces cerevisiae is the common microbes employed in ethanol production due to its high ethanol productivity, high ethanol tolerance and ability of fermenting wide range of sugars. However, there are some challenges in yeast fermentation which inhibit ethanol production such as high temperature, high ethanol concentration and the ability to ferment pentose sugars. Various types of yeast strains have been used in fermentation for ethanol production including hybrid, recombinant and wild-type yeasts. Yeasts can directly ferment simple sugars into ethanol while other type of feedstocks must be converted to fermentable sugars before it can be fermented to ethanol. The common processes involves in ethanol production are pretreatment, hydrolysis and fermentation. Production of bioethanol during fermentation depends on several factors such as temperature, sugar concentration, pH, fermentation time, agitation rate, and inoculum size. The efficiency and productivity of ethanol can be enhanced by immobilizing the yeast cells. This review highlights the different types of yeast strains, fermentation process, factors affecting bioethanol production and immobilization of yeasts for better bioethanol production.

Effects of yeast stress and pH on 3-monochloropropanediol (3-MCPD)-producing reactions in model dough systems.

PubMed

Hamlet, C G; Sadd, P A

2005-07-01

A major precursor of 3-monochloropropanediol (3-MCPD) in leavened cereal products is glycerol, which is formed as a natural by-product of yeast fermentation. However, yeast metabolism is affected by stresses such as low osmotic pressure from, for example, the incorporation of sugar or salt in the dough recipe. Tests with model doughs have shown that glycerol production was proportional to yeast mass and limited by available sugars, but that high levels of yeast inhibited 3-MCPD formation. The yeast fraction responsible for the inhibition of 3-MCPD in model dough was shown to be the soluble cytosol proteins, and the inhibition mechanism could be explained by the known reactions of 3-MCPD and/or its precursors with ammonia/amino acids (from yeast proteins). Added glucose did not increase the production of glycerol by yeast but it did promote the generation of 3-MCPD in cooked doughs. The latter effect was attributed to the removal of 3-MCPD inhibitors such as ammonia and amino acids by their reactions with added glucose (e.g. Maillard). The thermal generation of organic acids from added glucose also reduced the pH of cooked doughs, so the effect of pH and short-chain organic acids on 3-MCPD generation in dough was measured. There was a good correlation between initial dough pH and the level of 3-MCPD generated. The effect was weaker than that predicted by simple kinetic modelling, suggesting that the involvement of H+ and/or the organic acid was catalytic. The results showed that modifications to dough recipes involving the addition of reducing sugars and/or organic acids can have a significant impact on 3-MPCD generation in bakery products.

Aqueous chemical growth of alpha-Fe2O3-alpha-Cr203 nanocompositethin films

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vayssieres, Lionel; Guo, Jinghua; Nordgren, Joseph

2001-06-30

We are reporting here on the inexpensive fabrication and optical properties of an iron(III) oxide chromium(III) oxide nanocomposite thin film of corundum crystal structure. Its novel and unique-designed architecture consists of uniformed, well-defined and oriented nanorods of Hematite (alpha-Fe2O3) of 50 nm in diameter and 500nm in length and homogeneously distributed nonaggregated monodisperse spherical nanoparticles of Eskolaite (alpha-Cr2O3) of 250 nm in diameter. This alpha-Fe2O3 alpha-Cr2O3 nanocomposite thin film is obtained by growing, directly onto transparent polycrystalline conducting substrate, an oriented layer of hematite nanorods and growing subsequently, the eskolaite layer. The synthesis is carried out by a template-free, low-temperature,more » multilayer thin film coating process using aqueous solution of metal salts as precursors. Almost 100 percent of the light is absorbed by the composite film between 300 and 525 nm and 40 percent at 800 nm which yields great expectations as photoanode materials for photovoltaic cells and photocatalytic devices.« less

Comparison of dry sheet media and conventional agar media methods for enumerating yeasts and molds in food.

PubMed

Beuchat, L R; Mann, David A; Gurtler, Joshua B

2007-11-01

A study was done to compare Nissui Compact Dry Yeast and Mold plates (CDYM), 3M Petrifilm Yeast and Mold count plates (PYM), dichloran-rose bengal chloramphenicol (DRBC) agar, and dichloran 18% glycerol (DG18) agar for enumerating yeasts and molds naturally occurring in 97 foods (grains, legumes, raw fruits and vegetables, nuts, dairy products, meats, and miscellaneous processed foods and dry mixes). Correlation coefficients for plates incubated for 5 days were DG18 versus DRBC (0.93), PYM versus DRBC (0.81), CDYM versus DG18 (0.81), PYM versus DG18 (0.80), CDYM versus DRBC (0.79), and CDYM versus PYM (0.75). The number of yeasts and molds recovered from a group of foods (n = 32) analyzed on a weight basis (CFU per gram) was not significantly different (alpha = 0.05) when samples were plated on DRBC, DG18, PYM, or CDYM. However, the order of recovery from foods (n = 65) in a group analyzed on a unit or piece basis, or a composite of both groups (n = 97), was DRBC > DG18 = CDYM > PYM. Compared with PYM, CDYM recovered equivalent, significantly higher (alpha = 0.05) or significantly lower (alpha = 0.05) numbers of yeasts and molds in 51.5, 27.8, and 20.6%, respectively, of the 97 foods tested; respective values were 68.8, 15.6, and 15.6% in the small group (n = 32) and 43.1, 33.8, and 23.1% in the large group (n = 65) of foods. The two groups contained different types of foods, the latter consisting largely (73.8%) of raw fruits (n = 16) and vegetables (n = 32). Differences in efficacy of the four methods in recovering yeasts and molds from foods in the two groups are attributed in part to differences in genera and predominant mycoflora. While DG18 agar, CDYM, and PYM appear to be acceptable for enumerating yeasts and molds in the foods analyzed in this study, overall, DRBC agar recovered higher numbers from the 97 test foods, thereby supporting its recommended use as a general purpose medium for mycological analysis.

Hepatocyte nuclear factor-4alpha is a central transactivator of the mouse Ntcp gene.

PubMed

Geier, Andreas; Martin, Ina V; Dietrich, Christoph G; Balasubramaniyan, Natarajan; Strauch, Sonja; Suchy, Frederick J; Gartung, Carsten; Trautwein, Christian; Ananthanarayanan, Meenakshisundaram

2008-08-01

Sodium taurocholate cotransporting polypeptide (Ntcp) is the major uptake system for conjugated bile acids. Deletions of hepatocyte nuclear factor (HNF)-1alpha and retinoid X receptor-alpha:retinoic acid receptor-alpha binding sites in the mouse 5'-flanking region corresponding to putatively central regulatory elements of rat Ntcp do not significantly reduce promoter activity. We hypothesized that HNF-4alpha, which is increasingly recognized as a central regulator of hepatocyte function, may directly transactivate mouse (mNtcp). A 1.1-kb 5'-upstream region including the mouse Ntcp promoter was cloned and compared with the rat promoter. In contrast to a moderate 3.5-fold activation of mNtcp by HNF-1alpha, HNF-4alpha cotransfection led to a robust 20-fold activation. Deletion analysis of mouse and rat Ntcp promoters mapped a conserved HNF-4alpha consensus site at -345/-326 and -335/-316 bp, respectively. p-475bpmNtcpLUC is not transactivated by HNF-1alpha but shows a 50-fold enhanced activity upon cotransfection with HNF-4alpha. Gel mobility shift assays demonstrated a complex of the HNF-4alpha-element formed with liver nuclear extracts that was blocked by an HNF-4alpha specific antibody. HNF-4alpha binding was confirmed by chromatin immunoprecipitation. Using Hepa 1-6 cells, HNF-4alpha-knockdown resulted in a significant 95% reduction in NTCP mRNA. In conclusion, mouse Ntcp is regulated by HNF-4alpha via a conserved distal cis-element independently of HNF-1alpha.

Cuprophane but not synthetic membrane induces increases in serum tumor necrosis factor-alpha levels during hemodialysis.

PubMed

Canivet, E; Lavaud, S; Wong, T; Guenounou, M; Willemin, J C; Potron, G; Chanard, J

1994-01-01

Cytokine synthesis and secretion by blood mononuclear cells is a well-documented phenomenon in hemodialyzed patients. The present study was conducted in 17 chronically hemodialyzed patients to test the relative effect of uremic toxicity, membrane biocompatibility, dialysate composition, and the risk of endotoxinemia on the serum level of tumor necrosis factor-alpha (TNF-alpha). The only significant parameter that influenced circulating TNF-alpha was the chemical characteristics of the dialyzer membrane. Tumor necrosis factor-alpha levels significantly increased during the session with cuprophane, whereas they decreased with AN69. The TNF-alpha increase was documented whatever the dialysate buffer and the presence or absence (negative Limulus amoebocyte lysate test) of endotoxin in the dialysate. In the subgroup of patients treated with a contaminated dialysate and AN69, none had clinical symptoms and the central body temperature remained constant throughout the session. In these patients, serum TNF-alpha levels did not change after priming the dialyzer with sterile saline. In conclusion, the serum TNF-alpha level during hemodialysis appears to be modulated by biocompatibility, permeability, and binding properties of dialysis membrane rather than dialysate composition. Endotoxin in the dialysate did not result in positive TNF-alpha balance no matter what its possible priming effect on mononucleated blood cells.

The Leu72Met Polymorphism of the Prepro-ghrelin Gene is Associated With Alcohol Consumption and Subjective Responses to Alcohol: Preliminary Findings

PubMed Central

Suchankova, Petra; Yan, Jia; Schwandt, Melanie L.; Stangl, Bethany L.; Jerlhag, Elisabet; Engel, Jörgen A.; Hodgkinson, Colin A.; Ramchandani, Vijay A.; Leggio, Lorenzo

2017-01-01

Abstract Aims The orexigenic peptide ghrelin may enhance the incentive value of food-, drug- and alcohol-related rewards. Consistent with preclinical findings, human studies indicate a role of ghrelin in alcohol use disorders (AUD). In the present study an a priori hypothesis-driven analysis was conducted to investigate whether a Leu72Met missense polymorphism (rs696217) in the prepro-ghrelin gene (GHRL), is associated with AUD, alcohol consumption and subjective responses to alcohol. Method Association analysis was performed using the National Institute on Alcohol Abuse and Alcoholism (NIAAA) clinical sample, comprising AUD individuals and controls (N = 1127). Then, a post-hoc analysis using data from a human laboratory study of intravenous alcohol self-administration (IV-ASA, N = 144) was performed to investigate the association of this SNP with subjective responses following a fixed dose of alcohol (priming phase) and alcohol self-administration (ad libitum phase). Results The case-control study revealed a trend association (N = 1127, OR = 0.665, CI = 0.44–1.01, P = 0.056) between AUD diagnosis and Leu72Met. In AUD subjects, the SNP was associated with significantly lower average drinks per day (n = 567, β = −2.49, 95% CI = −4.34 to −0.64, P = 0.008) and significantly fewer heavy drinking days (n = 567, β = −12.00, 95% CI = −19.10 to −4.89, P < 0.001). The IV-ASA study further revealed that 72Met carriers had greater subjective responses to alcohol (P < 0.05) when compared to Leu72Leu both at priming and during ad lib self-administration. Conclusion Although preliminary, these findings suggest that the Leu72Leu genotype may lead to increased risk of AUD possibly via mechanisms involving a lower response to alcohol resulting in excessive alcohol consumption. Further investigations are warranted. Short Summary We investigated whether a Leu72Met missense polymorphism in the prepro-ghrelin gene, is associated with alcohol use disorder, alcohol

The Leu72Met Polymorphism of the Prepro-ghrelin Gene is Associated With Alcohol Consumption and Subjective Responses to Alcohol: Preliminary Findings.

PubMed

Suchankova, Petra; Yan, Jia; Schwandt, Melanie L; Stangl, Bethany L; Jerlhag, Elisabet; Engel, Jörgen A; Hodgkinson, Colin A; Ramchandani, Vijay A; Leggio, Lorenzo

2017-07-01

The orexigenic peptide ghrelin may enhance the incentive value of food-, drug- and alcohol-related rewards. Consistent with preclinical findings, human studies indicate a role of ghrelin in alcohol use disorders (AUD). In the present study an a priori hypothesis-driven analysis was conducted to investigate whether a Leu72Met missense polymorphism (rs696217) in the prepro-ghrelin gene (GHRL), is associated with AUD, alcohol consumption and subjective responses to alcohol. Association analysis was performed using the National Institute on Alcohol Abuse and Alcoholism (NIAAA) clinical sample, comprising AUD individuals and controls (N = 1127). Then, a post-hoc analysis using data from a human laboratory study of intravenous alcohol self-administration (IV-ASA, N = 144) was performed to investigate the association of this SNP with subjective responses following a fixed dose of alcohol (priming phase) and alcohol self-administration (ad libitum phase). The case-control study revealed a trend association (N = 1127, OR = 0.665, CI = 0.44-1.01, P = 0.056) between AUD diagnosis and Leu72Met. In AUD subjects, the SNP was associated with significantly lower average drinks per day (n = 567, β = -2.49, 95% CI = -4.34 to -0.64, P = 0.008) and significantly fewer heavy drinking days (n = 567, β = -12.00, 95% CI = -19.10 to -4.89, P < 0.001). The IV-ASA study further revealed that 72Met carriers had greater subjective responses to alcohol (P < 0.05) when compared to Leu72Leu both at priming and during ad lib self-administration. Although preliminary, these findings suggest that the Leu72Leu genotype may lead to increased risk of AUD possibly via mechanisms involving a lower response to alcohol resulting in excessive alcohol consumption. Further investigations are warranted. We investigated whether a Leu72Met missense polymorphism in the prepro-ghrelin gene, is associated with alcohol use disorder, alcohol consumption and subjective responses to alcohol. Although preliminary

Membrane-derived second messenger regulates x-ray-mediated tumor necrosis factor alpha gene induction.

PubMed Central

Hallahan, D E; Virudachalam, S; Kuchibhotla, J; Kufe, D W; Weichselbaum, R R

1994-01-01

Cells adapt to adverse environmental conditions through a wide range of responses that are conserved throughout evolution. Physical agents such as ionizing radiation are known to initiate a stress response that is triggered by the recognition of DNA damage. We have identified a signaling pathway involving the activation of phospholipase A2 and protein kinase C in human cells that confers x-ray induction of the tumor necrosis factor alpha gene. Treatment of human cells with ionizing radiation or H2O2 was associated with the production of arachidonic acid. Inhibition of phospholipase A2 abolished radiation-mediated arachidonate production as well as the subsequent activation of protein kinase C and tumor necrosis factor alpha gene expression. These findings demonstrate that ionizing radiation-mediated gene expression in human cells is regulated in part by extranuclear signal transduction. One practical application of phospholipase A2 inhibitors is to ameliorate the adverse effects of radiotherapy associated with tumor necrosis factor alpha production. Images PMID:8197153

Hepatocyte nuclear factor-4alpha induces transdifferentiation of hematopoietic cells into hepatocytes.

PubMed

Khurana, Satish; Jaiswal, Amit K; Mukhopadhyay, Asok

2010-02-12

Hematopoietic stem cells can directly transdifferentiate into hepatocytes because of cellular plasticity, but the molecular basis of transdifferentiation is not known. Here, we show the molecular basis using lineage-depleted oncostatin M receptor beta-expressing (Lin(-)OSMRbeta(+)) mouse bone marrow cells in a hepatic differentiation culture system. Differentiation of the cells was marked by the expression of albumin. Hepatocyte nuclear factor (HNF)-4alpha was expressed and translocated into the nuclei of the differentiating cells. Suppression of its activation in OSM-neutralized culture medium inhibited cellular differentiation. Ectopic expression of full-length HNF4alpha in 32D myeloid cells resulted in decreased myeloid colony-forming potential and increased expression of hepatocyte-specific genes and proteins. Nevertheless, the neohepatocytes produced in culture expressed active P450 enzyme. The obligatory role of HNF4alpha in hepatic differentiation was confirmed by transfecting Lin(-)OSMRbeta(+) cells with dominant negative HNF4alpha in the differentiation culture because its expression inhibited the transcription of the albumin and tyrosine aminotransferase genes. The loss and gain of functional activities strongly suggested that HNF4alpha plays a central role in the transdifferentiation process. For the first time, this report demonstrates the mechanism of transdifferentiation of hematopoietic cells into hepatocytes, in which HNF4alpha serves as a molecular switch.

Tumor necrosis factor-alpha stimulates the production of squamous cell carcinoma antigen in normal squamous cells.

PubMed

Numa, F; Takeda, O; Nakata, M; Nawata, S; Tsunaga, N; Hirabayashi, K; Suminami, Y; Kato, H; Hamanaka, S

1996-01-01

Squamous cell carcinoma (SCC) antigen, a tumor marker of squamous cell carcinoma, is also increased in several nonmalignant skin lesions, e.g. pemphigus. The aim of the present investigation was to determine if tumor necrosis factor-alpha (TNF-alpha), one of the important environmental factors, stimulated the production of SCC antigen in the normal squamous cells. The exposure of normal human epidermal keratinocytes to TNF-alpha (100 IU/ml) for 72 h greatly increased the SCC antigen production. The stimulatory effect of TNF-alpha (1,000 IU/ml) on the production of SCC antigen was also observed in the normal squamous epithelium tissue. These results would be helpful for understanding the increase of SCC antigen in several nonmalignant skin disorders.

Effects of asparagine, fructose, and baking conditions on acrylamide content in yeast-leavened wheat bread.

PubMed

Surdyk, Nicolas; Rosén, Johan; Andersson, Roger; Aman, Per

2004-04-07

A repeatable procedure for studying the effects of internal and external factors on acrylamide content in yeast-leavened wheat bread has been developed. The dough contained wheat endosperm flour with a low content of precursors for acrylamide formation (asparagine and reducing sugars), dry yeast, salt, and water. The effects of asparagine and fructose, added to the dough, were studied in an experiment with a full factorial design. More than 99% of the acrylamide was found in the crust. Added asparagine dramatically increased the content of acrylamide in crusts dry matter (from about 80 microg/kg to between 600 and 6000 microg/kg) while added fructose did not influence the content. The effects of temperature and time of baking were studied in another experiment using a circumscribed central composite design. Mainly temperature (above 200 degrees C) but also time increased the acrylamide content in crust dry matter (from below 10 to 1900 microg/kg), and a significant interaction was found between these two factors. When baked at different conditions with the same ingredients, a highly significant relationship (P < 0.001) between color and acrylamide content in crust was found. Added asparagine, however, did not increase color, showing that mainly other amino compounds are involved in the browning reactions.

Opportunistic Pathogenic Yeasts

NASA Astrophysics Data System (ADS)

Banerjee, Uma

Advances in medical research, made during the last few decades, have improved the prophylactic, diagnostic and therapeutic capabilities for variety of infections/diseases. However, many of the prophylactic and therapeutic procedures have been seen in many instances to exact a price of host-vulnerability to an expanding group of opportunistic pathogens and yeasts are one of the important members in it. Fortunately amongst the vast majority of yeasts present in nature only few are considered to have the capability to cause infections when certain opportunities predisposes and these are termed as ‘opportunistic pathogenic yeasts.’ However, the term ‘pathogenic’ is quite tricky, as it depends of various factors of the host, the ‘bug’ and the environment to manifest the clinical infection. The borderline is expanding. In the present century with unprecedented increase in number of immune-compromised host in various disciplines of health care settings, where any yeast, which has the capability to grow at 37 ° C (normal body temperature of human), can be pathogenic and cause infection in particular situation

Thalidomide in rat liver cirrhosis: blockade of tumor necrosis factor-alpha via inhibition of degradation of an inhibitor of nuclear factor-kappaB.

PubMed

Paul, Shelley Chireyath; Lv, Peng; Xiao, Yan-Jv; An, Ping; Liu, Shi-Quan; Luo, He-Sheng

2006-01-01

Thalidomide inhibited tumor necrosis factor-alpha (TNF-alpha) effectively in many trials. The aim of this study was to investigate the effect of thalidomide on the expression of nuclear factor-kappaB (NF-kappaB), inhibitor of NF-kappaB (IkappaB) and TNF-alpha in a rat model of liver cirrhosis. Liver cirrhosis was achieved by intraperitoneal injection of carbon tetrachloride thrice weekly, and thalidomide (10 or 100 mg/kg/day) was given daily by intragastric route for 8 weeks. Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), prealbumin (PA), hyaluronic acid (HA) and laminin (LN), and hydroxyproline (HYP), NF-kappaBp65, alpha-smooth muscle actin (alpha-SMA) protein and TNF-alpha mRNA were studied in the liver, IkappaBalpha and TNF-alpha protein in the cytoplasm and NF-kappaBp65 protein in the nucleus. Compared with nontreated cirrhotic rats, the histopathology of rats given thalidomide (100 mg/kg) was significantly better. Serum ALT, AST, HA and LN and HYP content in the liver were significantly decreased and PA was elevated (p < 0.01) in this group; the expression of TNF-alpha mRNA and protein, NF-kappaBp65 and alpha-SMA were significantly decreased and IkappaBalpha protein was also elevated (p < 0.01). Thalidomide downregulates NF-kappaB-induced TNF-alpha and activates hepatic stellate cells (HSC) via inhibition of IkappaB degradation to prevent liver cirrhosis. Copyright 2006 S. Karger AG, Basel.

HNF4alpha dysfunction as a molecular rational for cyclosporine induced hypertension.

PubMed

Niehof, Monika; Borlak, Jürgen

2011-01-27

Induction of tolerance against grafted organs is achieved by the immunosuppressive agent cyclosporine, a prominent member of the calcineurin inhibitors. Unfortunately, its lifetime use is associated with hypertension and nephrotoxicity. Several mechanism for cyclosporine induced hypertension have been proposed, i.e. activation of the sympathetic nervous system, endothelin-mediated systemic vasoconstriction, impaired vasodilatation secondary to reduction in prostaglandin and nitric oxide, altered cytosolic calcium translocation, and activation of the renin-angiotensin system (RAS). In this regard the molecular basis for undue RAS activation and an increased signaling of the vasoactive oligopeptide angiotensin II (AngII) remain elusive. Notably, angiotensinogen (AGT) is the precursor of AngII and transcriptional regulation of AGT is controlled by the hepatic nuclear factor HNF4alpha. To better understand the molecular events associated with cyclosporine induced hypertension, we investigated the effect of cyclosporine on HNF4alpha expression and activity and searched for novel HNF4alpha target genes among members of the RAS cascade. Using bioinformatic algorithm and EMSA bandshift assays we identified angiotensin II receptor type 1 (AGTR1), angiotensin I converting enzyme (ACE), and angiotensin I converting enzyme 2 (ACE2) as genes targeted by HNF4alpha. Notably, cyclosporine represses HNF4alpha gene and protein expression and its DNA-binding activity at consensus sequences to AGT, AGTR1, ACE, and ACE2. Consequently, the gene expression of AGT, AGTR1, and ACE2 was significantly reduced as evidenced by quantitative real-time RT-PCR. While RAS is composed of a sophisticated interplay between multiple factors we propose a decrease of ACE2 to enforce AngII signaling via AGTR1 to ultimately result in vasoconstriction and hypertension. Taken collectively we demonstrate cyclosporine to repress HNF4alpha activity through calcineurin inhibitor mediated inhibition of nuclear

Signalling mechanism for somatostatin receptor 5-mediated suppression of AMPA responses in rat retinal ganglion cells.

PubMed

Deng, Qin-Qin; Sheng, Wen-Long; Zhang, Gong; Weng, Shi-Jun; Yang, Xiong-Li; Zhong, Yong-Mei

2016-08-01

Somatostatin (SRIF) is involved in a variety of physiological functions via the activation of five subtypes of specific receptors (sst1-5). Here, we investigated the effects of SRIF on AMPA receptor (AMPAR)-mediated currents (AMPA currents) in isolated rat retinal ganglion cells (GCs) using patch-clamp techniques. Immunofluorescence double labelling demonstrated the expression of sst5 in rat GCs. Consistent to this, whole cell AMPA currents of GCs were dose-dependently suppressed by SRIF, and the effect was reversed by the sst5 antagonist BIM-23056. Intracellular dialysis of GDP-β-S or pre-incubation with the Gi/o inhibitor pertussis toxin (PTX) abolished the SRIF effect. The SRIF effect was mimicked by the administration of either 8-Br-cAMP or forskolin, but was eliminated by the protein kinase A (PKA) antagonists H-89/KT5720/Rp-cAMP. Moreover, SRIF increased intracellular Ca(2+) levels and did not suppress the AMPA currents when GCs were infused with an intracellular Ca(2+)-free solution or in the presence of ryanodine receptor modulators caffeine/ryanodine. Furthermore, the SRIF effect was eliminated when the activity of calmodulin (CaM), calcineurin and protein phosphatase 1 (PP1) was blocked with W-7, FK-506 and okadaic acid, respectively. SRIF persisted to suppress the AMPA currents when cGMP-protein kinase G (PKG) and phosphatidylinositol (PI)-/phosphatidylcholine (PC)-phospholipase C (PLC) signalling pathways were blocked. In rat flat-mount retinas, SRIF suppressed AMPAR-mediated light-evoked excitatory postsynaptic currents (L-EPSCs) in GCs. We conclude that a distinct Gi/o/cAMP-PKA/ryanodine/Ca(2+)/CaM/calcineurin/PP1 signalling pathway comes into play due to the activation of sst5 to mediate the SRIF effect on GCs. Copyright © 2016 Elsevier Ltd. All rights reserved.

Further evidence from functional studies for somatostatin receptor heterogeneity in guinea-pig isolated ileum, vas deferens and right atrium.

PubMed Central

Feniuk, W.; Dimech, J.; Jarvie, E. M.; Humphrey, P. P.

1995-01-01

1. Somatostatin (SRIF) causes a concentration-dependent inhibition of neurotransmission in guinea-pig ileum and vas deferens as well as negative inotropy in guinea-pig isolated right atrium. The SRIF receptors mediating these effects have now been further characterized by use of the peptides BIM-23027, BIM-23056 and L-362855, reported as selective for the recombinant SRIF receptor types, sst2, sst3 and sst5, respectively. 2. BIM-23027 was a highly potent agonist at causing an inhibition of neurotransmission in the guinea-pig ileum (EC50 value 1.9 nM), being about 3 times more potent than SRIF (EC50 value 6.8 nM). In contrast, in both guinea-pig vas deferens and right atrial preparations, BIM-23027 was a relatively weak agonist being at least 30-100 times weaker than SRIF. In guinea-pig atria, BIM-23027 (3 microM) antagonized the negative inotropic action of SRIF28 (apparent pKB = 5.9 +/- 0.1) but had no effect on the negative inotropic action of cyclohexyladenosine. 3. The inhibitory effect of BIM-23027 in the guinea-pig ileum was readily desensitized. Prior exposure to BIM-23027 (0.3 microM) markedly attenuated the inhibitory effect of SRIF but had no effect on the inhibitory action of clonidine suggesting that BIM-23027 and SRIF act via a common receptor mechanism. 4. L-362855 caused a concentration-dependent inhibition of neurotransmission in both the guinea-pig ileum and vas deferens as well as causing negative inotropy in the guinea-pig atrium but was at least 30-100 times weaker than SRIF. In guinea-pig isolated atria, L-362855 (3 microM) did not antagonize the negative inotropic action of SRIF28.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7582529

Amyloid Precursor Protein and Alpha Synuclein Translation, Implications for Iron and Inflammation in Neurodegenerative diseases

PubMed Central

Cahill, Catherine M.; Lahiri, Debomoy K.; Huang, Xudong; Rogers, Jack T.

2014-01-01

Summary Recent studies that alleles in the hemochromatosis gene may accelerate the onset of Alzheimer's disease (AD) by five years have validated interest in the model in which metals (particularly iron) accelerate disease course. Biochemical and biophysical measurements demonstrated the presence of elevated levels of neurotoxic copper, zinc and iron in the brains of AD patients. Intracellular levels of amyloid precursor protein (APP) holoprotein were shown to be modulated via iron by a mechanism that is similar to the translation control of the ferritin L- and H mRNAs by Iron-responsive Element (IRE) RNA stem loops in their 5′untranslated regions (5′UTRs). Recently, we reported a putative IRE-like sequence to be present in the 5′UTR of the Parkinson's disease (PD) specific alpha synuclein (ASYN) transcript. ASYN encodes the non-Aβ component (NAC) of amyloid plaques. The demonstration of iron-dependent translation of APP mRNA, the involvement of metals in the plaque of AD patients and of increased iron in striatal neurons in the Substantia nigra (SN) of PD patients, have each encouraged the development of metal attenuating agents and iron chelators as a major new therapeutic strategy for the treatment of these neurodegenerative diseases. In the case of AD, metal based therapeutics may ultimately prove more cost effective than the use of an amyloid vaccine as the preferred anti-amyloid therapeutic strategy to ameliorate the cognitive decline of AD patients. PMID:19166904

Effect of elicitors and precursors on azadirachtin production in hairy root culture of Azadirachta indica.

PubMed

Srivastava, Smita; Srivastava, A K

2014-02-01

The present study involved strategies for enhancement in in vitro azadirachtin (commercially used biopesticide) production by hairy root cultivation of Azadirachta indica. Improvement in the azadirachtin production via triggering its biosynthetic pathway in plant cells was carried out by the exogenous addition of precursors and elicitors in the growth medium. Among the different abiotic stress inducers (Ag(+), Hg(+2), Co(+2), Cu(+2)) and signal molecules (methyl jasmonate and salicylic acid) tested, salicylic acid at 15 mg l(-1) of concentration was found to enhance the azadirachtin yield in the hairy roots to the maximum (up to 4.95 mg g(-1)). Similarly, among the different biotic elicitors tested (filter-sterilized fungal culture filtrates of Phoma herbarium, Alternaria alternata, Myrothecium sp., Fusarium solani, Curvularia lunata, and Sclerotium rolfsii; yeast extract; and yeast extract carbohydrate fraction), addition of filter-sterilized fungal culture filtrate of C. lunata (1 % v/v) resulted in maximum azadirachtin yield enhancement in hairy root biomass (up to 7.1 mg g(-1)) with respect to the control (3.3 mg g(-1)). Among all the biosynthetic precursors studied (sodium acetate, cholesterol, squalene, isopentynyl pyrophosphate, mavalonic acid lactone, and geranyl pyrophosphate), the overall azadirachtin production (70.42 mg l(-1) in 25 days) was found to be the highest with cholesterol (50 mg l(-1)) addition as an indirect precursor in the medium.

Effect of salivary gland adenocarcinoma cell-derived alpha-N-acetylgalactosaminidase on the bioactivity of macrophage activating factor.

PubMed

Matsuura, Takashi; Uematsu, Takashi; Yamaoka, Minoru; Furusawa, Kiyofumi

2004-03-01

The aim of this study was to clarify the effects of alpha-N-acetylgalactosaminidase (alpha-NaGalase) produced by human salivary gland adenocarcinoma (SGA) cells on the bioactivity of macrophage-activating factor (GcMAF). High exo-alpha-NaGalase activity was detected in the SGA cell line HSG. HSG alpha-NaGalase had both exo- and endo-enzyme activities, cleaving the Gal-GalNAc and GalNAc residues linked to Thr/Ser but not releasing the [NeuAc2-6]GalNac residue. Furthermore, GcMAF enzymatically prepared from the Gc protein enhanced the superoxide-generation capacity and phagocytic activity of monocytes/macrophages. However, GcMAF treated with purified alpha-NaGalase did not exhibit these effects. Thus, HSG possesses the capacity to produce larger quantities of alpha-NaGalase, which inactivates GcMAF produced from Gc protein, resulting in reduced phagocytic activity and superoxide-generation capacity of monocytes/macrophages. The present data strongly suggest that HSG alpha-NaGalase acts as an immunodeficiency factor in cancer patients.

The TWIST1 oncogene is a direct target of hypoxia-inducible factor-2alpha.

PubMed

Gort, E H; van Haaften, G; Verlaan, I; Groot, A J; Plasterk, R H A; Shvarts, A; Suijkerbuijk, K P M; van Laar, T; van der Wall, E; Raman, V; van Diest, P J; Tijsterman, M; Vooijs, M

2008-03-06

Hypoxia-inducible factors (HIFs) are highly conserved transcription factors that play a crucial role in oxygen homeostasis. Intratumoral hypoxia and genetic alterations lead to HIF activity, which is a hallmark of solid cancer and is associated with poor clinical outcome. HIF activity is regulated by an evolutionary conserved mechanism involving oxygen-dependent HIFalpha protein degradation. To identify novel components of the HIF pathway, we performed a genome-wide RNA interference screen in Caenorhabditis elegans, to suppress HIF-dependent phenotypes, like egg-laying defects and hypoxia survival. In addition to hif-1 (HIFalpha) and aha-1 (HIFbeta), we identified hlh-8, gska-3 and spe-8. The hlh-8 gene is homologous to the human oncogene TWIST1. We show that TWIST1 expression in human cancer cells is enhanced by hypoxia in a HIF-2alpha-dependent manner. Furthermore, intronic hypoxia response elements of TWIST1 are regulated by HIF-2alpha, but not HIF-1alpha. These results identify TWIST1 as a direct target gene of HIF-2alpha, which may provide insight into the acquired metastatic capacity of hypoxic tumors.

Pituitary transcription factor Prop-1 stimulates porcine pituitary glycoprotein hormone alpha subunit gene expression.

PubMed

Sato, Takanobu; Kitahara, Kousuke; Susa, Takao; Kato, Takako; Kato, Yukio

2006-10-01

Recently, we have reported that a Prophet of Pit-1 homeodomain factor, Prop-1, is a novel transcription factor for the porcine follicle-stimulating hormone beta subunit (FSHbeta) gene. This study subsequently aimed to examine the role of Prop-1 in the gene expression of two other porcine gonadotropin subunits, pituitary glycoprotein hormone alpha subunit (alphaGSU), and luteinizing hormone beta subunit (LHbeta). A series of deletion mutants of the porcine alphaGSU (up to -1059 bp) and LHbeta (up to -1277 bp) promoters were constructed in the reporter vector, fused with the secreted alkaline phosphatase gene (pSEAP2-Basic). Transient transfection studies using GH3 cells were carried out to estimate the activation of the porcine alphaGSU and LHbeta promoters by Prop-1, which was found to activate the alphaGSU promoter of -1059/+12 bp up to 11.7-fold but not the LHbeta promoter. Electrophoretic mobility shift assay and DNase I footprinting analysis revealed that Prop-1 binds to six positions, -1038/-1026, -942/-928, -495/-479, -338/-326, -153/-146, and -131/-124 bp, that comprise the A/T cluster. Oligonucleotides of six Prop-1 binding sites were directly connected to the minimum promoter of alphaGSU, fused in the pSEAP2-Basic vector, followed by transfecting GH3 cells to determine the cis-acting activity. Finally, we concluded that at least five Prop-1 binding sites are the cis-acting elements for alphaGSU gene expression. The present results revealed a notable feature of the proximal region, where three Prop-1-binding sites are close to and/or overlap the pituitary glycoprotein hormone basal element, GATA-binding element, and junctional regulatory element. To our knowledge, this is the first demonstration of the role of Prop-1 in the regulation of alphaGSU gene expression. These results, taken together with our previous finding that Prop-1 is a transcription factor for FSHbeta gene, confirm that Prop-1 modulates the synthesis of FSH at the transcriptional level. On

Effects of cysteamine administration on the in vivo incorporation of (/sup 35/S)cysteine into somatostatin-14, somatostatin-28, arginine vasopressin, and oxytocin in rat hypothalamus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cameron, J.L.; Fernstrom, J.D.

1986-09-01

The effect of cysteamine injection on the in vivo incorporation of (/sup 35/S)cysteine into somatostatin-14 (SRIF-14), SRIF-28, arginine vasopressin (AVP), and oxytocin (OXT) in rat hypothalamus was studied. (/sup 35/S)Cysteine was injected into the third ventricle 1 h, 4 h, or 1 week after cysteamine (300 mg/kg, sc) injection; animals were killed 4 h later. The drug was found to substantially reduce immunoreactive SRIF levels, but not OXT or AVP, 4 h after its injection. Cysteamine also caused large reductions in label incorporation into SRIF-14, SRIF-28, and OXT 1 and 4 h after drug injection. However, (/sup 35/S)cysteine incorporation intomore » AVP was increased substantially at these time points, while that into acid-precipitable protein was normal. One week after cysteamine injection, label incorporation into all hypothalamic peptides was normal. Cysteine specific activity was also measured after (/sup 35/S)cysteine injection and was found to be similar in treatment and control groups. The results suggest that cysteamine inhibits the syntheses of SRIF-14, SRIF-28, and OXT and stimulates that of AVP.« less

Syphilis in the Setting of Anti-tumor Necrosis Factor Alpha Therapy.

PubMed

Iglesias-Plaza, Ana; Iglesias-Sancho, Maribel; Quintana-Codina, Mónica; García-Miguel, Javier; Salleras-Redonnet, Montse

2018-02-03

Inhibitors of tumor necrosis factor-alpha (anti-TNF-alpha) are widely used in different medical specialties. The main adverse effect of these agents is the increased risk of infection. We report the case of a 30-year-old man with ankylosing spondylitis who had begun receiving golimumab two weeks earlier. He presented with a 10-day history of salmon-colored lesions on trunk, palms and soles. The clinical suspicion was secondary syphilis. Treponemal and nontreponemal tests confirmed the diagnosis of syphilis. Lumbar puncture was also performed, although there was no neurological involvement, to rule out neurosyphilis. Cases of syphilis in patients in treatment with TNF-alpha inhibitors are uncommon in the literature and there are no established protocols. Copyright © 2017 Elsevier España, S.L.U. and Sociedad Española de Reumatología y Colegio Mexicano de Reumatología. All rights reserved.

Aromatic hydrocarbon biodegradation activates neutral lipid biosynthesis in oleaginous yeast.

PubMed

Deeba, Farha; Pruthi, Vikas; Negi, Yuvraj S

2018-05-01

In this study, the biodegradation ability of oleaginous yeast Cryptococcus psychrotolerans IITRFD for aromatic hydrocarbons (AHs) was investigated. It was found to completely degrade range of AHs such as 1 g/L phenol, 0.75 g/L naphthalene, 0.50 g/L anthracene and 0.50 g/L pyrene with lipid productivity (g/L/h) of 0.0444, 0.0441, 0.0394 and 0.0383, respectively. This work demonstrated the ring cleavage pathways of AHs by this yeast which follow ortho route for phenol and naphthalene while meta route for anthracene and pyrene degradation. The end products generated during biodegradation of AHs are feed as precursors for de novo triacylglycerols (TAG) biosynthesis pathway of oleaginous yeast. A high quantity of lipid content (46.54%) was observed on phenol as compared to lipid content on naphthalene (46.38%), anthracene (44.97%) and pyrene (44.16%). The lipid profile revealed by GC-MS analysis shows elevated monounsaturated fatty acid (MUFA) content with improved biodiesel quality. Copyright © 2018 Elsevier Ltd. All rights reserved.

Pro-region engineering for improved yeast display and secretion of brain derived neurotrophic factor.

PubMed

Burns, Michael L; Malott, Thomas M; Metcalf, Kevin J; Puguh, Arthya; Chan, Jonah R; Shusta, Eric V

2016-03-01

Brain derived neurotrophic factor (BDNF) is a promising therapeutic candidate for a variety of neurological diseases. However, it is difficult to produce as a recombinant protein. In its native mammalian context, BDNF is first produced as a pro-protein with subsequent proteolytic removal of the pro-region to yield mature BDNF protein. Therefore, in an attempt to improve yeast as a host for heterologous BDNF production, the BDNF pro-region was first evaluated for its effects on BDNF surface display and secretion. Addition of the wild-type pro-region to yeast BDNF production constructs improved BDNF folding both as a surface-displayed and secreted protein in terms of binding its natural receptors TrkB and p75, but titers remained low. Looking to further enhance the chaperone-like functions provided by the pro-region, two rounds of directed evolution were performed, yielding mutated pro-regions that further improved the display and secretion properties of BDNF. Subsequent optimization of the protease recognition site was used to control whether the produced protein was in pro- or mature BDNF forms. Taken together, we have demonstrated an effective strategy for improving BDNF compatibility with yeast protein engineering and secretion platforms. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Elevated serum tumor necrosis factor-alpha and soluble tumor necrosis factor receptors correlate with aberrant energy metabolism in liver cirrhosis.

PubMed

Shiraki, Makoto; Terakura, Yoichi; Iwasa, Junpei; Shimizu, Masahito; Miwa, Yoshiyuki; Murakami, Nobuo; Nagaki, Masahito; Moriwaki, Hisataka

2010-03-01

Protein-energy malnutrition is frequently observed in patients with liver cirrhosis and is associated with their poor prognosis. Tumor necrosis factor-alpha (TNF-alpha) is elevated in those patients and may contribute to the alterations of energy metabolism. Our aim was to characterize the aberrant energy metabolism in cirrhotic patients with regard to TNF-alpha. Twenty-four patients (mean age 65 +/- 6 y) with viral liver cirrhosis who did not have hepatocellular carcinoma or acute infections were studied. Twelve healthy volunteers were recruited after matching for age, gender, and body mass index with the patients and served as controls (59 +/- 8 y). Serum levels of TNF-alpha, soluble 55-kDa TNF receptor (sTNF-R55), soluble 75-kDa TNF receptor (sTNF-R75), and leptin were determined by immunoassay. Substrate oxidation rates of carbohydrate and fat were estimated by indirect calorimetry after overnight bedrest and fasting. In cirrhotic patients, serum levels of TNF-alpha, sTNF-R55, and sTNF-R75 were significantly higher than those in the controls and correlated with the increasing grade of disease severity as defined by Child-Pugh classification. Serum leptin concentration was not different between cirrhotics and controls but correlated with their body mass index. The decrease in substrate oxidation rate of carbohydrate and the increase in substrate oxidation rate of fat significantly correlated with serum TNF-alpha, sTNF-R55, and sTNF-R75 concentrations. Tumor necrosis factor-alpha might be associated with the aberrant energy metabolism in patients with liver cirrhosis. Copyright (c) 2010 Elsevier Inc. All rights reserved.

Bioflavoring by non-conventional yeasts in sequential beer fermentations.

PubMed

Holt, Sylvester; Mukherjee, Vaskar; Lievens, Bart; Verstrepen, Kevin J; Thevelein, Johan M

2018-06-01

Non-conventional yeast species have great capacity for producing diverse flavor profiles in production of alcoholic beverages, but their potential for beer brewing, in particular in consecutive fermentations with Saccharomyces cerevisiae, has only poorly been explored. We have screened 17 non-conventional yeast species for production of an appealing profile of flavor esters and phenolics in the first phase of alcoholic fermentation, followed by inoculation with S. cerevisiae to complete the fermentation. For measurement of phenolic compounds and their precursors we developed an improved and highly sensitive methodology. The results show that non-conventional yeast species possess promising potential for enhancement of desirable flavors in beer production. Notable examples are increasing isoamyl acetate (fruity, banana flavor) by application of P. kluyverii, augmenting ethyl phenolic compounds (spicy notes) with Brettanomyces species and enhancing 4-vinyl guaiacol (clove-like aroma) with T. delbrueckii. All Pichia strains also produced high levels of ethyl acetate (solvent-like flavor). This might be selectively counteracted by selection of an appropriate S. cerevisiae strain for the second fermentation phase, which lowers total ester profile. Hence, optimization of the process conditions and/or proper strain selection in sequentially inoculated fermentations are required to unlock the full potential for aroma improvement by the non-conventional yeast species. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

The 53-kDa proteolytic product of precursor starch-hydrolyzing enzyme of Aspergillus niger has Taka-amylase-like activity.

PubMed

Ravi-Kumar, K; Venkatesh, K S; Umesh-Kumar, S

2007-04-01

The 53-kDa amylase secreted by Aspergillus niger due to proteolytic processing of the precursor starch-hydrolyzing enzyme was resistant to acarbose, a potent alpha-glucosidase inhibitor. The enzyme production was induced when A. niger was grown in starch medium containing the inhibitor. Antibodies against the precursor enzyme cross-reacted with the 54-kDa Taka-amylase protein of A. oryzae. It resembled Taka-amylase in most of its properties and also hydrolyzed starch to maltose of alpha-anomeric configuration. However, it did not degrade maltotriose formed during the reaction and was not inhibited by zinc ions.

Platelet factor XIII increases the fibrinolytic resistance of platelet-rich clots by accelerating the crosslinking of alpha 2-antiplasmin to fibrin

NASA Technical Reports Server (NTRS)

Reed, G. L.; Matsueda, G. R.; Haber, E.

1992-01-01

Platelet clots resist fibrinolysis by plasminogen activators. We hypothesized that platelet factor XIII may enhance the fibrinolytic resistance of platelet-rich clots by catalyzing the crosslinking of alpha 2-antiplasmin (alpha 2AP) to fibrin. Analysis of plasma clot structure by polyacrylamide gel electrophoresis and immunoblotting revealed accelerated alpha 2AP-fibrin crosslinking in platelet-rich compared with platelet-depleted plasma clots. A similar study of clots formed with purified fibrinogen (depleted of factor XIII activity), isolated platelets, and specific factor XIII inhibitors indicated that this accelerated crosslinking was due to the catalytic activity of platelet factor XIII. Moreover, when washed platelets were aggregated by thrombin, there was evidence of platelet factor XIII-mediated crosslinking between platelet alpha 2AP and platelet fibrin(ogen). Specific inhibition (by a monoclonal antibody) of the alpha 2AP associated with washed platelet aggregates accelerated the fibrinolysis of the platelet aggregate. Thus in platelet-rich plasma clots, and in thrombin-induced platelet aggregates, platelet factor XIII actively formed alpha 2AP-fibrin crosslinks, which appeared to enhance the resistance of platelet-rich clots to fibrinolysis.

Identification of a novel cyclosporin-sensitive element in the human tumor necrosis factor alpha gene promoter

PubMed Central

1993-01-01

Tumor necrosis factor alpha (TNF-alpha), a cytokine with pleiotropic biological effects, is produced by a variety of cell types in response to induction by diverse stimuli. In this paper, TNF-alpha mRNA is shown to be highly induced in a murine T cell clone by stimulation with T cell receptor (TCR) ligands or by calcium ionophores alone. Induction is rapid, does not require de novo protein synthesis, and is completely blocked by the immunosuppressant cyclosporin A (CsA). We have identified a human TNF-alpha promoter element, kappa 3, which plays a key role in the calcium-mediated inducibility and CsA sensitivity of the gene. In electrophoretic mobility shift assays, an oligonucleotide containing kappa 3 forms two DNA protein complexes with proteins that are present in extracts from unstimulated T cells. These complexes appear in nuclear extracts only after T cell stimulation. Induction of the inducible nuclear complexes is rapid, independent of protein synthesis, and blocked by CsA, and thus, exactly parallels the induction of TNF-alpha mRNA by TCR ligands or by calcium ionophore. Our studies indicate that the kappa 3 binding factor resembles the preexisting component of nuclear factor of activated T cells. Thus, the TNF-alpha gene is an immediate early gene in activated T cells and provides a new model system in which to study CsA-sensitive gene induction in activated T cells. PMID:8376940

Tracking Diacylglycerol and Phosphatidic Acid Pools in Budding Yeast

PubMed Central

Ganesan, Suriakarthiga; Shabits, Brittney N.; Zaremberg, Vanina

2015-01-01

Phosphatidic acid (PA) and diacylglycerol (DAG) are key signaling molecules and important precursors for the biosynthesis of all glycerolipids found in eukaryotes. Research conducted in the model organism Saccharomyces cerevisiae has been at the forefront of the identification of the enzymes involved in the metabolism and transport of PA and DAG. Both these lipids can alter the local physical properties of membranes by introducing negative curvature, but the anionic nature of the phosphomonoester headgroup in PA sets it apart from DAG. As a result, the mechanisms underlying PA and DAG interaction with other lipids and proteins are notoriously different. This is apparent from the analysis of the protein domains responsible for recognition and binding to each of these lipids. We review the current evidence obtained using the PA-binding proteins and domains fused to fluorescent proteins for in vivo tracking of PA pools in yeast. In addition, we present original results for visualization of DAG pools in yeast using the C1 domain from mammalian PKCδ. An emerging first cellular map of the distribution of PA and DAG pools in actively growing yeast is discussed. PMID:27081314

Nicergoline stimulates protein kinase C mediated alpha-secretase processing of the amyloid precursor protein in cultured human neuroblastoma SH-SY5Y cells.

PubMed

Cedazo-Minguez, A; Bonecchi, L; Winblad, B; Post, C; Wong, E H; Cowburn, R F; Benatti, L

1999-10-01

We investigated the ability of the antidementia agents, nicergoline, aniracetam and hydergine to stimulate PKC mediated alpha-secretase amyloid precursor protein (APP) processing in cultured human neuroblastoma SH-SY5Y cells. Western immunoblotting of cell conditioned media using the Mabs 22C11 and 6E10 revealed the presence of 2 bands with molecular mass of 90 and 120 kDa, corresponding to possible alternatively glycosylated forms of secreted APP (APPs). Short-term (30 min and 2 h) treatment of cells with nicergoline gave an increased intensity of both bands, compared to non-treated cells. Maximal nicergoline effects, of the order of 150-200% over basal APPs release, were seen at concentrations between 1 and 10 microM. Under the same condition, 1 microM PdBu, used as a positive control, gave 500-1000% increases of basal APPs release. In contrast, aniracetam and hydergine, did not show any effect on APPs secretion. 2 h treatment with nicergoline had no effect on cellular full-length APP levels, as determined by immunoblotting of cell extracts with 22C11 and CT15 antibodies. Immunoblotting with PKC isoform specific antibodies of soluble and membrane fractions prepared from 2 h treated cells, showed that nicergoline (50 microM) and PdBu (1 microM) both induced translocation of PKC alpha, gamma and epsilon, but not PKC beta. The involvement of PKC in mediating nicergoline stimulated APPs release was also studied using specific inhibitors. 1 microM calphostin C, a broad range PKC inhibitor, significantly reduced both PdBu (1 microM) and nicergoline (10 microM) induced APPs release. In contrast, Go6976 (1 microM), a selective PKC alpha and beta1 inhibitor, as well as the cAMP-dependent protein kinase inhibitor, H89 (1 microM) were without effect. These results indicate that nicergoline can modulate alpha-secretase APP processing by a PKC dependent mechanism that is likely to involve the gamma and epsilon isoforms of this enzyme.

TERATOGENIC RESPONSES ARE MODULATED IN MICE LACKING EXPRESSION OF EPIDERMAL GROWTH FACTOR (EGF) AND TRANSFORMING GROWTH FACTOR-ALPHA (TGF)

EPA Science Inventory

TITLE:
TERATOGENIC RESPONSES ARE MODULATED IN MICE LACKING EXPRESSION OF EPIDERMAL GROWTH FACTOR (EGF) AND TRANSFORMING GROWTH FACTOR-ALPHA (TGF). AUTHORS (ALL): Abbott, Barbara D.1; Best, Deborah S.1; Narotsky, Michael G.1. SPONSOR NAME: None INSTITUTIONS (ALL): 1. Repro Tox ...

Identification of the antigenic determinants of factors 8, 9, and 34 of genus Candida.

PubMed

Kobayashi, H; Oyamada, H; Suzuki, A; Shibata, N; Suzuki, S; Okawa, Y

1996-10-21

We investigated the antigenic determinants of factors 8, 9, and 34 of the genus Candida among pathogenic yeasts by enzyme-linked immunosorbent assay (ELISA) using mannans of Saccharomyces cerevisiae wild type and mutant types, mnn 1-mnn 4 and mnn 2. Results of ELISA including antisera against the antigenic factors of genus Candida (Candida Check, latron; FAbs) indicated that these three types of mannan distinctly react with FAbs 34, 8 and 9, respectively. To identify the recognition sites of these FAbs, we compared the ability of various oligosaccharides to inhibit the binding of the mannans to FAbs. The results indicated that FAb 34 preferentially recognizes linear side chains containing a non-reducing terminal alpha-1,3-linked mannose residue, Man(alpha)1 --> 3Man(alpha)1 --> (2Man(alpha)1 --> )n(2Man) (n > or = 0), and that one of the recognition sites of FAb 9 is linear alpha-1,6-linked oligomannosyl series, Man(alpha)1 --> (6Man(alpha)1 --> )n(6Man) (n > or = 2). On the other hand, the recognition site of FAb 8 apparently consisted of two alpha-1,2-linked oligomannosyl side chains and an alpha-1,6-linked mannose residue that originated from the mannan backbone, Man(alpha)1 --> 2Man(alpha)1 --> 2(Man(alpha)1 -->2Man(alpha)1 --> 6)Man.

Tumor Necrosis Factor-Alpha and Interleukin 6 in Human Periapical Lesions

PubMed Central

Pršo, Ivana Brekalo; Kocjan, Willy; Šimić, Hrvoje; Brumini, Gordana; Pezelj-Ribarić, Sonja; Borčić, Josipa; Ferreri, Silvio; Karlović, Ivana Miletić

2007-01-01

Aim. The aim of this study was to evaluate the presence of the cytokines tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) in human periapical lesions. Subjects and methods. Samples were obtained from three groups of teeth: symptomatic teeth, asymptomatic lesions, and uninflamed periradicular tissues as a control. Results. TNF-alpha levels were significantly increased in symptomatic lesions compared to control. Group with asymptomatic lesions had significantly higher concentrations compared to control. There were no significant differences in TNF-alpha levels between symptomatic and asymptomatic lesions. In group with symptomatic lesions, IL-6 levels were significantly higher than in group with asymptomatic lesions. The IL-6 levels in symptomatic group also showed significantly higher concentration in comparison with control group. In asymptomatic group, the IL-6 level had significantly higher concentrations compared to control. Conclusion. These results indicate that symptomatic lesions represent an immunologically active stage of disease, and asymptomatic lesions are the point from which the process advances toward healing. PMID:17497030

[The yeast biofilm in human medicine].

PubMed

Růzicka, Filip; Holá, Veronika; Votava, Miroslav

2007-08-01

In recent years, the role of Candida yeasts as causative agents of nosocomial infections has increased. One of the important virulence factors contributing to the development of such infections is biofilm production. This virulence factor enables yeast to colonize both native surfaces and artificial implants. The most common sources of infection are patients themselves, in particular the gastrointestinal tract and skin. The vectors of exogenous yeast infections are predominantly the hands of the health personnel and contaminated medical instruments. The adhesion of yeasts to the implant surfaces is determined both by implant surface and yeast characteristics. This is followed by proliferation and production of microcolonies and extracellular matrix. The final biofilm structure is also influenced by the production of hyphae and pseudohyphae. The entire process of biofilm production is controlled by numerous regulatory systems, with the key role being played by the quorum sensing system. Like the adhered bacterial cultures, candidas growing in the form of a biofilm are highly resistant to antimicrobial therapy. Resistance of yeast biofilms to antifungals is a complex process with multiple contributing factors. These are especially increased gene expression (e.g. genes encoding the so called multidrug efflux pumps), limited penetration of substances through the extracellular matrix, inhibited cell growth and altered microenvironment in deeper biofilm layers. The concentrations of antifungals able to effectively affect the biofilm cells exceed, by several orders of magnitude, the values of conventionally determined MICs. High biofilm resistance results in ineffective antifungal therapy of biofilm infections. Therefore, if possible, the colonized implant should be removed. Conservative therapy should involve antifungals with a proven effect on the biofilm (e.g. caspofungin). The most effective measure in fighting biofilm infections is prevention, especially adhering to

The role of co-opted ESCRT proteins and lipid factors in protection of tombusviral double-stranded RNA replication intermediate against reconstituted RNAi in yeast

PubMed Central

Nagy, Peter D.

2017-01-01

Reconstituted antiviral defense pathway in surrogate host yeast is used as an intracellular probe to further our understanding of virus-host interactions and the role of co-opted host factors in formation of membrane-bound viral replicase complexes in protection of the viral RNA against ribonucleases. The inhibitory effect of the RNA interference (RNAi) machinery of S. castellii, which only consists of the two-component DCR1 and AGO1 genes, was measured against tomato bushy stunt virus (TBSV) in wild type and mutant yeasts. We show that deletion of the co-opted ESCRT-I (endosomal sorting complexes required for transport I) or ESCRT-III factors makes TBSV replication more sensitive to the RNAi machinery in yeast. Moreover, the lack of these pro-viral cellular factors in cell-free extracts (CFEs) used for in vitro assembly of the TBSV replicase results in destruction of dsRNA replication intermediate by a ribonuclease at the 60 min time point when the CFE from wt yeast has provided protection for dsRNA. In addition, we demonstrate that co-opted oxysterol-binding proteins and membrane contact sites, which are involved in enrichment of sterols within the tombusvirus replication compartment, are required for protection of viral dsRNA. We also show that phosphatidylethanolamine level influences the formation of RNAi-resistant replication compartment. In the absence of peroxisomes in pex3Δ yeast, TBSV subverts the ER membranes, which provide as good protection for TBSV dsRNA against RNAi or ribonucleases as the peroxisomal membranes in wt yeast. Altogether, these results demonstrate that co-opted protein factors and usurped lipids are exploited by tombusviruses to build protective subcellular environment against the RNAi machinery and possibly other cellular ribonucleases. PMID:28759634

Thalidomide inhibits lipopolysaccharide-induced tumor necrosis factor-alpha production via down-regulation of MyD88 expression.

PubMed

Noman, Abu Shadat M; Koide, Naoki; Hassan, Ferdaus; I-E-Khuda, Imtiaz; Dagvadorj, Jargalsaikhan; Tumurkhuu, Gantsetseg; Islam, Shamima; Naiki, Yoshikazu; Yoshida, Tomoaki; Yokochi, Takashi

2009-02-01

The effect of thalidomide on lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF)-alpha production was studied by using RAW 264.7 murine macrophage-like cells. Thalidomide significantly inhibited LPS-induced TNF-alpha production. Thalidomide prevented the activation of nuclear factor (NF)-KB by down-regulating phosphorylation of inhibitory KB factor (IKB), and IKB kinase (IKK)-alpha and IKK-beta Moreover, thalidomide inhibited LPS-induced phosphorylation of AKT, p38 and stress-activated protein kinase (SAPK)/JNK. The expression of myeloid differentiation factor 88 (MyD88) protein and mRNA was markedly reduced in thalidomide-treated RAW 264.7 cells but there was no significant alteration in the expression of interleukin-1 receptor-associated kinase (IRAK) 1 and TNF receptor-associated factor (TRAF) 6 in the cells. Thalidomide did not affect the cell surface expression of Toll-like receptor (TLR) 4 and CD14, suggesting the impairment of intracellular LPS signalling in thalidomide-treated RAW 264.7 cells. Thalidomide significantly inhibited the TNF-alpha production in response to palmitoyl-Cys(RS)-2,3-di(palmitoyloxy) propyl)-Ala-Gly-OH (Pam(3)Cys) as a MyD88-dependent TLR2 ligand. Therefore, it is suggested that thalidomide might impair LPS signalling via down-regulation of MyD88 protein and mRNA and inhibit LPS-induced TNF-alpha production. The putative mechanism of thalidomide-induced MyD88 down-regulation is discussed.

Role of tumor necrosis factor-alpha and platelet-activating factor in neoangiogenesis induced by synovial fluids of patients with rheumatoid arthritis.

PubMed

Lupia, E; Montrucchio, G; Battaglia, E; Modena, V; Camussi, G

1996-08-01

The aim of the present study was to investigate in vivo in a mouse model the stimulation of neoangiogenesis by synovial fluids of patients with rheumatoid arthritis (RA) and to determine the role of tumor necrosis factor (TNF)-alpha and platelet-activating factor (PAF) in the formation of new vessels. Angiogenesis was studied in a mouse model in which Matrigel, injected subcutaneously, was used as a vehicle for the delivery of potential angiogenic stimuli. Synovial fluids of patients with RA but not with osteoarthritis (OA) were shown to induce neoangiogenesis. Since synovial fluid of patients with RA contained significantly higher levels of TNF-alpha-like bioactivity and of PAF than that of patients with OA, the role of these mediators was evaluated by using an anti-TNF-alpha neutralizing monoclonal antibody (mAb) and a PAF receptor antagonist, WEB 2170. When added to Matrigel, anti-TNF-alpha mAb and particularly WEB 2170 significantly reduced neoangiogenesis induced by synovial fluids of RA patients. Moreover, PAF extracted and purified from synovial fluid induced angiogenesis. These results suggest that the neoangiogenesis observed in rheumatoid synovitis may be due, at least in part, to the angiogenic effect of locally produced TNF-alpha and PAF.

The lager yeast Saccharomyces pastorianus removes and transforms Fusarium trichothecene mycotoxins during fermentation of brewer's wort.

PubMed

Nathanail, Alexis V; Gibson, Brian; Han, Li; Peltonen, Kimmo; Ollilainen, Velimatti; Jestoi, Marika; Laitila, Arja

2016-07-15

An investigation was conducted to determine the fate of deoxynivalenol, deoxynivalenol-3-glucoside, HT-2 toxin and T-2 toxin, during a four-day fermentation with the lager yeast Saccharomyces pastorianus. The influence of excessive mycotoxin concentrations on yeast growth, productivity and viability were also assessed. Mycotoxins were dosed at varying concentrations to 11.5° Plato wort. Analysis of yeast revealed that presence of the toxins even at concentrations up to 10,000 μg/L had little or no effect on sugar utilisation, alcohol production, pH, yeast growth or cell viability. Of the dosed toxin amounts 9-34% were removed by the end of fermentation, due to physical binding and/or biotransformation by yeast. Deoxynivalenol-3-glucoside was not reverted to its toxic precursor during fermentation. Processing of full-scan liquid chromatography-quadrupole time-of-flight-mass spectrometry (LC-QTOF-MS) data with MetaboLynx and subsequent LC-QTOF-MS/MS measurements resulted in annotation of several putative metabolites. De(acetylation), glucosylation and sulfonation were the main metabolic pathways activated. Copyright © 2016 Elsevier Ltd. All rights reserved.

Mutagenizing brewing yeast strain for improving fermentation property of beer.

PubMed

Liu, Zengran; Zhang, Guangyi; Sun, Yunping

2008-07-01

A brewing yeast mutant with perfect sugar fermentation capacity was isolated by mutagenizing the Saccharomyces pastorianus transformant, which carries an integrated glucoamylase gene and has one copy of non-functional alpha-acetolactate synthase gene. The mutant was able to utilize maltotriose efficiently, and the maltotriose fermentability in YNB-2% maltotriose medium increased from 32.4% to 72.0% after 5 d in shaking culture. The wort fermentation test confirmed that the sugar fermentation property of the mutant was greatly improved, while its brewing performances were analogous to that of the wild-type strain and the characteristic trait of shortened beer maturation period was retained. Therefore, we believe that the brewing yeast mutant would benefit the beer industry and would be useful for low caloric beer production.

Degradation of spent craft brewer’s yeast by caprine rumen hyper ammonia-producing bacteria

USDA-ARS?s Scientific Manuscript database

Spent brewer’s yeast has long been included in ruminant diets as a protein supplement. However, modern craft beers often include more hops (Humulus lupulus L.) compounds than traditional recipes. These compounds include alpha and beta-acids, which are antimicrobial to the rumen hyper ammonia-produci...

Alpha-cluster preformation factor within cluster-formation model for odd-A and odd-odd heavy nuclei

NASA Astrophysics Data System (ADS)

Saleh Ahmed, Saad M.

2017-06-01

The alpha-cluster probability that represents the preformation of alpha particle in alpha-decay nuclei was determined for high-intensity alpha-decay mode odd-A and odd-odd heavy nuclei, 82 < Z < 114, 111 < N < 174. This probability was calculated using the energy-dependent formula derived from the formulation of clusterisation states representation (CSR) and the hypothesised cluster-formation model (CFM) as in our previous work. Our previous successful determination of phenomenological values of alpha-cluster preformation factors for even-even nuclei motivated us to expand the work to cover other types of nuclei. The formation energy of interior alpha cluster needed to be derived for the different nuclear systems with considering the unpaired-nucleon effect. The results showed the phenomenological value of alpha preformation probability and reflected the unpaired nucleon effect and the magic and sub-magic effects in nuclei. These results and their analyses presented are very useful for future work concerning the calculation of the alpha decay constants and the progress of its theory.

Activation of protein kinase C by mycobacterial cord factor, trehalose 6-monomycolate, resulting in tumor necrosis factor-alpha release in mouse lung tissues.

PubMed

Sueoka, E; Nishiwaki, S; Okabe, S; Iida, N; Suganuma, M; Yano, I; Aoki, K; Fujiki, H

1995-08-01

Cord factors are mycoloyl glycolipids in cell walls of bacteria belonging to Actinomycetales, such as Mycobacterium, Nocardia and Rhodococcus. They induce granuloma formation in the lung and interstitial pneumonitis, associated with production of macrophage-derived cytokines. We studied how cord factors induce biological activities in the cells. Cord factors isolated from M. tuberculosis, trehalose 6-monomycolate (mTMM) and trehalose 6,6'-dimycolate (mTDM), enhanced protein kinase C (PKC) activation in the presence of phosphatidylserine (PtdSer), diacylglycerol and Ca2+, and mTMM activated PKC alpha more strongly than PKC beta or gamma under the same assay conditions. Kinetic studies of mTMM in response to PKC activation revealed that mTMM increased the apparent affinity of PKC to Ca2+ in the presence of both PtdSer and diolein. Although this is similar to observations with unsaturated fatty acids, such as arachidonic acid, mTMM was synergistic with PtdSer for PKC activation, but arachidonic acid was not. mTMM was also different as regards PKC activation, as phorbol ester was. A single i.p. administration of mTMM to mouse induced tumor necrosis factor-alpha (TNF-alpha) in serum and in the lung, which is a unique target tissue of cord factors. Based on our recent finding that TNF-alpha is an endogenous tumor promoter, the correlation between lung cancer and pulmonary tuberculosis is discussed.

Overexpression of hypoxia-inducible factor 1 alpha improves immunomodulation by dental mesenchymal stem cells.

PubMed

Martinez, Victor G; Ontoria-Oviedo, Imelda; Ricardo, Carolina P; Harding, Sian E; Sacedon, Rosa; Varas, Alberto; Zapata, Agustin; Sepulveda, Pilar; Vicente, Angeles

2017-09-29

Human dental mesenchymal stem cells (MSCs) are considered as highly accessible and attractive MSCs for use in regenerative medicine, yet some of their features are not as well characterized as other MSCs. Hypoxia-preconditioning and hypoxia-inducible factor 1 (HIF-1) alpha overexpression significantly improves MSC therapeutics, but the mechanisms involved are not fully understood. In the present study, we characterize immunomodulatory properties of dental MSCs and determine changes in their ability to modulate adaptive and innate immune populations after HIF-1 alpha overexpression. Human dental MSCs were stably transduced with green fluorescent protein (GFP-MSCs) or GFP-HIF-1 alpha lentivirus vectors (HIF-MSCs). A hypoxic-like metabolic profile was confirmed by mitochondrial and glycolysis stress test. Capacity of HIF-MSCs to modulate T-cell activation, dendritic cell differentiation, monocyte migration, and polarizations towards macrophages and natural killer (NK) cell lytic activity was assessed by a number of functional assays in co-cultures. The expression of relevant factors were determined by polymerase chain reaction (PCR) analysis and enzyme-linked immunosorbent assay (ELISA). While HIF-1 alpha overexpression did not modify the inhibition of T-cell activation by MSCs, HIF-MSCs impaired dendritic cell differentiation more efficiently. In addition, HIF-MSCs showed a tendency to induce higher attraction of monocytes, which differentiate into suppressor macrophages, and exhibited enhanced resistance to NK cell-mediated lysis, which supports the improved therapeutic capacity of HIF-MSCs. HIF-MSCs also displayed a pro-angiogenic profile characterized by increased expression of CXCL12/SDF1 and CCL5/RANTES and complete loss of CXCL10/IP10 transcription. Immunomodulation and expression of trophic factors by dental MSCs make them perfect candidates for cell therapy. Overexpression of HIF-1 alpha enhances these features and increases their resistance to allogenic NK

Association of the macrophage activating factor (MAF) precursor activity with polymorphism in vitamin D-binding protein.

PubMed

Nagasawa, Hideko; Sasaki, Hideyuki; Uto, Yoshihiro; Kubo, Shinichi; Hori, Hitoshi

2004-01-01

Serum vitamin D-binding protein (Gc protein or DBP) is a highly expressed polymorphic protein, which is a precursor of the inflammation-primed macrophage activating factor, GcMAF, by a cascade of carbohydrate processing reactions. In order to elucidate the relationship between Gc polymorphism and GcMAF precursor activity, we estimated the phagocytic ability of three homotypes of Gc protein, Gc1F-1F, Gc1S-1S and Gc2-2, through processing of their carbohydrate moiety. We performed Gc typing of human serum samples by isoelectric focusing (IEF). Gc protein from human serum was purified by affinity chromatography with 25-hydroxyvitamin D3-sepharose. A phagocytosis assay of Gc proteins, modified using beta-glycosidase and sialidase, was carried out. The Gc1F-1F phenotype was revealed to possess Galbeta1-4GalNAc linkage by the analysis of GcMAF precursor activity using beta1-4 linkage-specific galactosidase from jack bean. The GcMAF precursor activity of the Gc1F-1F phenotype was highest among three Gc homotypes. The Gc polymorphism and carbohydrate diversity of Gc protein are significant for its pleiotropic effects.

[Rbf1 (RPG-box binding factor), a transcription factor involved in yeast-hyphal transition of Candida albicans].

PubMed

Aoki, Y; Ishii, N; Watanabe, M; Yoshihara, F; Arisawa, M

1998-01-01

The major fungal pathogen for fungal diseases which have become a major medical problem in the last few years is Candida albicans, which can grow both in yeast and hyphae forms. This ability of C. albicans is thought to contribute to its colonization and dissemination within host tissues. In a recent few years, accompanying the introduction of molecular biological tools into C. albicans organism, several factors involved in the signal transduction pathway for yeast-hyphal transition have been identified. One MAP kinase pathway in C. albicans, similar to that leading to STE12 activation in Saccharomyces cerevisiae, has been reported. C. albicans strains mutant in these genes show retarded filamentous growth on a solid media but no impairment of filamentous growth in mice. These results suggest two scenarios that a kinase signaling cascade plays a part in stimulating the morphological transition in C. albicans, and that there would be another signaling pathway effective in animals. In this latter true hyphal pathway, although some candidate proteins, such as Efg1 (transcription factor), Int1 (integrin-like membrane protein), or Phr1 (pH-regulated membrane protein), have been identified, it is still too early to say that we understand the whole picture of that cascade. We have cloned a C. albicans gene encoding a novel DNA binding protein, Rbf1, that predominantly localizes in the nucleus, and shows transcriptional activation capability. Disruption of the functional RBF1 genes of C. albicans induced the filamentous growth on all solid and liquid media tested, suggesting that Rbf1 might be another candidate for the true hyphal pathway. Relationships with other factors described above, and the target (regulated) genes of Rbf1 is under investigation.

Elevated tumour necrosis factor-alpha was associated with intima thickening in obese children.

PubMed

Bo, Luo; Yi-Can, Yang; Qing, Zhou; Xiao-Hui, Wu; Ke, Huang; Chao-Chun, Zou

2017-04-01

This study investigated the relationship between intima-media thickness (IMT) and immune parameters in obese children from five to 16 years of age. We enrolled 185 obese children with a mean age of 10.65 ± 2.10 years and 211 controls with a mean age of 10.32 ± 1.81 years. Glycometabolism, lipid metabolism, sex hormones, immune indices and carotid IMT were measured. Serum interleukin (IL)-6, IL-10, tumour necrosis factor (TNF)-alpha, white blood cells and common and internal carotid artery IMTs in the obese group were higher than those in the control group (p < 0.05, respectively). Bivariate correlation analysis showed that the common carotid arterial IMT was positively correlated with alanine aminotransferase, triglyceride, uric acid, apolipoprotein B, IL-6, IL-10, TNF-alpha, follicle-stimulating hormone and testosterone. Internal carotid artery IMT was positively correlated with alanine aminotransferase and follicle-stimulating hormone. Both common and internal carotid artery IMTs were inversely correlated with apolipoprotein A1 (p < 0.05, respectively). Stepwise multiple regression analysis showed that testosterone, alanine aminotransferase and TNF-alpha were the independent determinants of common carotid arterial IMT. Tumour necrosis factor-alpha, alanine aminotransferase and testosterone were associated with intima thickening in the early life in obese children and may increase later risks of premature atherogenicity and adult cardio-cerebrovascular diseases. ©2016 Foundation Acta Paediatrica. Published by John Wiley & Sons Ltd.

Induction of the nuclear factor HIF-1{alpha} in acetaminophen toxicity: Evidence for oxidative stress

DOE Office of Scientific and Technical Information (OSTI.GOV)

James, Laura P.; Donahower, Brian; Burke, Angela S.

2006-04-28

Hypoxia inducible factor (HIF) controls the transcription of genes involved in angiogenesis, erythropoiesis, glycolysis, and cell survival. HIF-1{alpha} levels are a critical determinant of HIF activity. The induction of HIF-1{alpha} was examined in the livers of mice treated with a toxic dose of APAP (300 mg/kg IP) and sacrificed at 1, 2, 4, 8, and 12 h. HIF-1{alpha} was induced at 1-12 h and induction occurred prior to the onset of toxicity. Pre-treatment of mice with N-acetylcysteine (1200 mg/kg IP) prevented toxicity and HIF-1{alpha} induction. In further studies, hepatocyte suspensions were incubated with APAP (1 mM) in the presence ofmore » an oxygen atmosphere. HIF-1{alpha} was induced at 1 h, prior to the onset of toxicity. Inclusion of cyclosporine A (10 {mu}M), an inhibitor of mitochondrial permeability transition, oxidative stress, and toxicity, prevented the induction of HIF-1{alpha}. Thus, HIF-1{alpha} is induced before APAP toxicity and can occur under non-hypoxic conditions. The data suggest a role for oxidative stress in the induction of HIF-1{alpha} in APAP toxicity.« less

Molecular cloning and expression analysis of sea bass (Dicentrarchus labrax L.) tumor necrosis factor-alpha (TNF-alpha).

PubMed

Nascimento, Diana S; Pereira, Pedro J B; Reis, Marta I R; do Vale, Ana; Zou, Jun; Silva, Manuel T; Secombes, Christopher J; dos Santos, Nuno M S

2007-09-01

In the search for pro-inflammatory genes in sea bass a TNF-alpha gene was cloned and sequenced. The sea bass TNF-alpha (sbTNF-alpha) putative protein conserves the TNF-alpha family signature, as well as the two cysteines usually involved in the formation of a disulfide bond. The mouse TNF-alpha Thr-Leu cleavage sequence and a potential transmembrane domain were also found, suggesting that sbTNF-alpha exists as two forms: a approximately 28 kDa membrane-bound form and a approximately 18.4 kDa soluble protein. The single copy sbTNF-alpha gene contains a four exon-three intron structure similar to other known TNF-alpha genes. Homology modeling of sbTNF-alpha is compatible with the trimeric quaternary architecture of its mammalian counterparts. SbTNF-alpha is constitutively expressed in several unstimulated tissues, and was not up-regulated in the spleen and head-kidney, in response to UV-killed Photobacterium damselae subsp. piscicida. However, an increase of sbTNF-alpha expression was detected in the head-kidney during an experimental infection using the same pathogen.

Effect of hypothalamic neuropeptides on corticotrophin release from quarters of rat anterior pituitary gland in vitro.

PubMed

Nicholson, S A; Adrian, T E; Gillham, B; Jones, M T; Bloom, S R

1984-02-01

The effect of six hypothalamic peptides on the basal release of ACTH and that induced by arginine vasopressin (AVP) or by ovine corticotrophin releasing factor (oCRF) from fragments of the rat anterior pituitary gland incubated in vitro was investigated. Dose-response curves to AVP and to oCRF were obtained, and the response to a low dose of oCRF was potentiated by a low dose of AVP. Basal release of ACTH was not affected by any of the peptides in concentrations in the range 10(-12) to 10(-6) mol/l, and only substance P (SP) and somatostatin (SRIF) inhibited significantly the response to oCRF in a dose-related manner. The responses to a range of doses of oCRF or AVP were reduced by 10(-8) and 10(-6) mol SP or SRIF/l, and to a greater extent by the higher dose. Except in the case of 10(-6) mol SRIF/l on the response to AVP, the response was not further diminished by preincubation of the tissue with the peptide before the stimulating agent was added. The inhibition of the responses to AVP or oCRF by 10(-9) mol SP/l was not potentiated by its combination with either 5 X 10(-10) or 10(-8) mol SRIF/l; the inhibitory effects were merely additive. The results suggest that although SRIF and SP are able to modulate the release of ACTH from the anterior pituitary gland, they do so only at a high concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

Formation by yeast of 2-furanmethanethiol and ethyl 2-mercaptopropionate aroma compounds in Japanese soy sauce.

PubMed

Meng, Qi; Hatakeyama, Makoto; Sugawara, Etsuko

2014-01-01

Two aroma compounds of volatile thiols, 2-furanmethanethiol (2FM) and ethyl 2-mercaptopropionate (ET2MP), were formed in five types of Japanese soy sauce during fermentation by yeast. The concentrations of 2FM and ET2MP in the soy sauce samples increased during alcoholic fermentation. The concentrations of 2FM and ET2MP were higher in the soy sauce fermented by Zygosaccharomyces rouxii than in that fermented by Candida versatilis. The enantiomers of ET2MP were separated by gas chromatography in a capillary column. The average enantiomeric ratio of ET2MP in the soy sauce was approximately 1:1. 2FM was formed by yeast in a medium prepared from cysteine and furfural, and cysteine is considered the key precursor of 2FM by yeast in soy sauce.

Localization and Characterization of alpha-Glucosidase Activity in Lactobacillus brevis.

PubMed

De Cort, S; Kumara, H M; Verachtert, H

1994-09-01

Lactobacillus brevis is found together with the yeast Brettanomyces lambicus during the overattenuation process in spontaneously fermented lambic beer. An isolated L. brevis strain has been shown to produce an alpha-glucosidase with many similarities to the glucosidase earlier found in B. lambicus. The enzyme was purified by ammonium sulfate precipitation, gel (Sephadex G-150 and Ultrogel AcA-44) filtration, and ion-exchange chromatography (DEAE-Sephadex A-50). The molecular weights of the enzyme, as determined by gel chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, were about 50,000 and 60,000, respectively. Optimum catalytic activity was obtained at 40 degrees C and pH 6.0. The enzyme showed a decrease of hydrolysis with an increase in the degree of polymerization of the substrate. The K(m) values for p-nitrophenyl-alpha-d-glucopyranoside, maltose, and maltotriose were 0.51, 3.0, and 5.2 mM, respectively. There was lack of inhibition by 0.15 mM acarbose and 0.5 M turanose, but the enzyme was inhibited by Tris (K(i) value of 25 mM). The alpha-glucosidase of L. brevis together with the enzyme of B. lambicus seems to be a key factor in the overattenuation of lambic beer, although the involvement of other lactic acid bacteria (pediococci) cannot be excluded.

XRN1 Is a Species-Specific Virus Restriction Factor in Yeasts

PubMed Central

Rowley, Paul A.; Ho, Brandon; Bushong, Sarah; Johnson, Arlen; Sawyer, Sara L.

2016-01-01

In eukaryotes, the degradation of cellular mRNAs is accomplished by Xrn1 and the cytoplasmic exosome. Because viral RNAs often lack canonical caps or poly-A tails, they can also be vulnerable to degradation by these host exonucleases. Yeast lack sophisticated mechanisms of innate and adaptive immunity, but do use RNA degradation as an antiviral defense mechanism. We find a highly refined, species-specific relationship between Xrn1p and the “L-A” totiviruses of different Saccharomyces yeast species. We show that the gene XRN1 has evolved rapidly under positive natural selection in Saccharomyces yeast, resulting in high levels of Xrn1p protein sequence divergence from one yeast species to the next. We also show that these sequence differences translate to differential interactions with the L-A virus, where Xrn1p from S. cerevisiae is most efficient at controlling the L-A virus that chronically infects S. cerevisiae, and Xrn1p from S. kudriavzevii is most efficient at controlling the L-A-like virus that we have discovered within S. kudriavzevii. All Xrn1p orthologs are equivalent in their interaction with another virus-like parasite, the Ty1 retrotransposon. Thus, Xrn1p appears to co-evolve with totiviruses to maintain its potent antiviral activity and limit viral propagation in Saccharomyces yeasts. We demonstrate that Xrn1p physically interacts with the Gag protein encoded by the L-A virus, suggesting a host-virus interaction that is more complicated than just Xrn1p-mediated nucleolytic digestion of viral RNAs. PMID:27711183

Physiological role of somatostatin-mediated autofeedback regulation for growth hormone: importance of growth hormone in triggering somatostatin release during a trough period of pulsatile growth hormone release in conscious male rats.

PubMed

Sato, M; Chihara, K; Kita, T; Kashio, Y; Okimura, Y; Kitajima, N; Fujita, T

1989-08-01

In mammals including human, it is generally accepted that growth hormone (GH) can regulate its own secretion through an autofeedback mechanism in which somatostatin (SRIF) may be involved. To explore a physiological role of SRIF-mediated GH autoregulation, the effect of exogenous human GH administration on plasma rat GH response to [D-Ala2, Nle27]-human GH-releasing hormone-(1-28)-agmatine (hGHRH-analog), which does not crossreact with anti-rat GH-releasing hormone gamma-globulin (GHRH-Ab), was examined in conscious male rats treated with GHRH-Ab in the absence and presence of anti-SRIF gamma-globulin (SRIF-Ab). Enhanced SRIF release during a trough period of natural pulsatile GH secretion, suggested by the blunted GH response to exogenous hGHRH-analog, no longer occurred when major GH secretory bursts were abolished by GHRH-Ab treatment. On the other hand, when hGH was administered in GHRH-Ab-treated rats so as to simulate the quantity and dynamic change of GH in hypophysial portal circulation in rats exhibiting pulsatile GH secretion, hGHRH-analog-induced GH rises were significantly suppressed during the period corresponding to a GH trough. This suppression was completely prevented by simultaneous treatment with SRIF-Ab. Furthermore, administration of bovine GH, but not ovine prolactin, resulted in significant suppression of hGHRH-analog-provoked GH rises. These findings suggest that enhanced SRIF release during a trough period of spontaneous GH secretory rhythm is induced by the preceding GH secretory burst, and also suggest a possible role for SRIF-mediated GH autoregulation in a physiological state.

SNPs Altering Ammonium Transport Activity of Human Rhesus Factors Characterized by a Yeast-Based Functional Assay

PubMed Central

Deschuyteneer, Aude; Boeckstaens, Mélanie; De Mees, Christelle; Van Vooren, Pascale; Wintjens, René; Marini, Anna Maria

2013-01-01

Proteins of the conserved Mep-Amt-Rh family, including mammalian Rhesus factors, mediate transmembrane ammonium transport. Ammonium is an important nitrogen source for the biosynthesis of amino acids but is also a metabolic waste product. Its disposal in urine plays a critical role in the regulation of the acid/base homeostasis, especially with an acid diet, a trait of Western countries. Ammonium accumulation above a certain concentration is however pathologic, the cytotoxicity causing fatal cerebral paralysis in acute cases. Alteration in ammonium transport via human Rh proteins could have clinical outcomes. We used a yeast-based expression assay to characterize human Rh variants resulting from non synonymous single nucleotide polymorphisms (nsSNPs) with known or unknown clinical phenotypes and assessed their ammonium transport efficiency, protein level, localization and potential trans-dominant impact. The HsRhAG variants (I61R, F65S) associated to overhydrated hereditary stomatocytosis (OHSt), a disease affecting erythrocytes, proved affected in intrinsic bidirectional ammonium transport. Moreover, this study reveals that the R202C variant of HsRhCG, the orthologue of mouse MmRhcg required for optimal urinary ammonium excretion and blood pH control, shows an impaired inherent ammonium transport activity. Urinary ammonium excretion was RHcg gene-dose dependent in mouse, highlighting MmRhcg as a limiting factor. HsRhCGR202C may confer susceptibility to disorders leading to metabolic acidosis for instance. Finally, the analogous R211C mutation in the yeast ScMep2 homologue also impaired intrinsic activity consistent with a conserved functional role of the preserved arginine residue. The yeast expression assay used here constitutes an inexpensive, fast and easy tool to screen nsSNPs reported by high throughput sequencing or individual cases for functional alterations in Rh factors revealing potential causal variants. PMID:23967154

Identification of the functional domain in the transcription factor RTEF-1 that mediates alpha 1-adrenergic signaling in hypertrophied cardiac myocytes.

PubMed

Ueyama, T; Zhu, C; Valenzuela, Y M; Suzow, J G; Stewart, A F

2000-06-09

Cardiac myocytes respond to alpha(1)-adrenergic receptor stimulation by a progressive hypertrophy accompanied by the activation of many fetal genes, including skeletal muscle alpha-actin. The skeletal muscle alpha-actin gene is activated by signaling through an MCAT element, the binding site of the transcription enhancer factor-1 (TEF-1) family of transcription factors. Previously, we showed that overexpression of the TEF-1-related factor (RTEF-1) increased the alpha(1)-adrenergic response of the skeletal muscle alpha-actin promoter, whereas TEF-1 overexpression did not. Here, we identified the functional domains and specific sequences in RTEF-1 that mediate the alpha(1)-adrenergic response. Chimeric TEF-1 and RTEF-1 expression constructs localized the region responsible for the alpha(1)-adrenergic response to the carboxyl-terminal domain of RTEF-1. Site-directed mutagenesis was used to inactivate eight serine residues of RTEF-1, not present in TEF-1, that are putative targets of alpha(1)-adrenergic-dependent kinases. Mutation of a single serine residue, Ser-322, reduced the alpha(1)-adrenergic activation of RTEF-1 by 70% without affecting protein stability, suggesting that phosphorylation at this serine residue accounts for most of the alpha(1)-adrenergic response. Thus, these results demonstrate that RTEF-1 is a direct target of alpha(1)-adrenergic signaling in hypertrophied cardiac myocytes.

Artificial ligand binding within the HIF2[alpha] PAS-B domain of the HIF2 transcription factor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Scheuermann, Thomas H.; Tomchick, Diana R.; Machius, Mischa

2009-05-12

The hypoxia-inducible factor (HIF) basic helix-loop-helix Per-aryl hydrocarbon receptor nuclear translocator (ARNT)-Sim (bHLH-PAS) transcription factors are master regulators of the conserved molecular mechanism by which metazoans sense and respond to reductions in local oxygen concentrations. In humans, HIF is critically important for the sustained growth and metastasis of solid tumors. Here, we describe crystal structures of the heterodimer formed by the C-terminal PAS domains from the HIF2{alpha} and ARNT subunits of the HIF2 transcription factor, both in the absence and presence of an artificial ligand. Unexpectedly, the HIF2{alpha} PAS-B domain contains a large internal cavity that accommodates ligands identified frommore » a small-molecule screen. Binding one of these ligands to HIF2{alpha} PAS-B modulates the affinity of the HIF2{alpha}:ARNT PAS-B heterodimer in vitro. Given the essential role of PAS domains in forming active HIF heterodimers, these results suggest a presently uncharacterized ligand-mediated mechanism for regulating HIF2 activity in endogenous and clinical settings.« less

Problem-Solving Test: Analysis of DNA Damage Recognizing Proteins in Yeast and Human Cells

ERIC Educational Resources Information Center

Szeberenyi, Jozsef

2013-01-01

The experiment described in this test was aimed at identifying DNA repair proteins in human and yeast cells. Terms to be familiar with before you start to solve the test: DNA repair, germline mutation, somatic mutation, inherited disease, cancer, restriction endonuclease, radioactive labeling, [alpha-[superscript 32]P]ATP, [gamma-[superscript…

Thalidomide inhibits tumor necrosis factor-alpha production and antigen presentation by Langerhans cells.

PubMed

Deng, Liang; Ding, Wanhong; Granstein, Richard D

2003-11-01

Thalidomide is an effective treatment for several inflammatory and autoimmune disorders including erythema nodosum leprosum, Behcet's syndrome, discoid lupus erythematosus, and Crohn's disease. Thalidomide is believed to exert its anti-inflammatory effects, at least in part, by inhibiting tumor necrosis factor-alpha (TNF-alpha) production by monocytes. We studied the effects of thalidomide on epidermal Langerhans cells (LC). LCs are epidermal antigen-presenting dendritic cells that play important roles in skin immune responses. Using the murine epidermis-derived dendritic cell lines, XS106A from A/J mice and XS52 from BALB/c mice as surrogates for LC, we found that thalidomide inhibited TNF-alpha production in a concentration-dependent manner. Northern blot analysis revealed that thalidomide significantly decreased the peak-induced mRNA level of TNF-alpha in XS106A cells and XS52 cells. We then examined the effect of thalidomide on fresh LC enriched to approximately 98% using positive selection of Ia+ cells with antibodies conjugated to magnetic microspheres. TNF-alpha production was reduced by 67.7% at a thalidomide concentration of 200 microg per mL. Thalidomide also had a profound inhibitory effect on the ability of LC to present antigen to a responsive TH1 clone. Thalidomide inhibits TNF-alpha production and the antigen-presenting ability of epidermal LCs. These mechanisms may contribute to the therapeutic effects observed with this agent.

Serum tumour necrosis factor alpha in osteopenic and osteoporotic postmenopausal females: A cross-sectional study in Pakistan.

PubMed

Murad, Rafat; Shezad, Zahra; Ahmed, Saara; Ashraf, Mussarat; Qadir, Murad; Rehman, Rehana

2018-03-01

To compare biochemical parameters serum tumour necrosis factor alpha, calcium, magnesium, bone-specific alkaline phosphatase and vitamin D in postmenopausal women. This cross-sectional study was carried out from June 2015 to July 2016 at Jinnah Medical and Dental College, Karachi, and comprised postmenopausal women. Bone mineral density done by dual energy X-ray absorptiometryscan categorised subjects by World Health Organisation classification into normal (T score > -1) osteopenic (T score between -1 and -2.5) and osteoporotic (T score < -2.5). Biochemical parameters like tumour necrosis alpha, calcium, magnesium, bone-specific alkaline phosphatase and vitamin D were measured by solid phase enzyme amplified sensitivity immunoassay method. SPSS 16 was used to analyse the data. Of the 146 women, 34(23%) were normal, 93(67%) were osteopenic and 19(13%) were osteoporotic. There was significant difference in mean body mass index, serum tumour necrosis factor alpha and calcium in all the three groups (p<0.01). Significant mean difference was observed in serum calcium levels between normal and osteopenic, and between normal and osteoporotic group (p<0.05 each) without any significant mean difference between osteopenic and osteoporotic groups (p>0.05). A significant difference was observed for mean tumour necrosis factor alpha values between normal and osteoporotic groups (p<0.05). Tumour necrosis factor alpha showed negative correlation with bone mineral density in osteopenic and osteoporotic groups (p>0.05). Increased bone turnover in postmenopausal osteopenic women can be predicted by increased serum cytokine.

Mobilization of steryl esters from lipid particles of the yeast Saccharomyces cerevisiae.

PubMed

Wagner, Andrea; Grillitsch, Karlheinz; Leitner, Erich; Daum, Günther

2009-02-01

In the yeast as in other eukaryotes, formation and hydrolysis of steryl esters (SE) are processes linked to lipid storage. In Saccharomyces cerevisiae, the three SE hydrolases Tgl1p, Yeh1p and Yeh2p contribute to SE mobilization from their site of storage, the lipid particles/droplets. Here, we provide evidence for enzymatic and cellular properties of these three hydrolytic enzymes. Using the respective single, double and triple deletion mutants and strains overexpressing the three enzymes, we demonstrate that each SE hydrolase exhibits certain substrate specificity. Interestingly, disturbance in SE mobilization also affects sterol biosynthesis in a type of feedback regulation. Sterol intermediates stored in SE and set free by SE hydrolases are recycled to the sterol biosynthetic pathway and converted to the final product, ergosterol. This recycling implies that the vast majority of sterol precursors are transported from lipid particles to the endoplasmic reticulum, where sterol biosynthesis is completed. Ergosterol formed through this route is then supplied to its subcellular destinations, especially the plasma membrane. Only a minor amount of sterol precursors are randomly distributed within the cell after cleavage from SE. Conclusively, SE storage and mobilization although being dispensable for yeast viability contribute markedly to sterol homeostasis and distribution.

Lipidomics in research on yeast membrane lipid homeostasis.

PubMed

de Kroon, Anton I P M

2017-08-01

Mass spectrometry is increasingly used in research on membrane lipid homeostasis, both in analyses of the steady state lipidome at the level of molecular lipid species, and in pulse-chase approaches employing stable isotope-labeled lipid precursors addressing the dynamics of lipid metabolism. Here my experience with, and view on mass spectrometry-based lipid analysis is presented, with emphasis on aspects of quantification of membrane lipid composition of the yeast Saccharomyces cerevisiae. This article is part of a Special Issue entitled: BBALIP_Lipidomics Opinion Articles edited by Sepp Kohlwein. Copyright © 2017 Elsevier B.V. All rights reserved.

Tumor necrosis factor alpha and pulmonary function in Saskatchewan grain handlers.

PubMed

McDuffie, Helen H; Nakagawa, Kazuko; Pahwa, Punam; Shindo, Junichi; Hashimoto, Mirai; Nakada, Naoyuki; Ghosh, Sunita; Kirychuk, Shelley P; Hucl, Pierre

2006-05-01

The objective of this study was to estimate the contribution of lifestyle (cigarettes) and tumor necrosis factor (TNF) alpha polymorphisms at position 308 of the tumor necrosis factor alpha gene promotor (TNF-308*1/*2) to pulmonary function among grain handlers. Employed male grain handlers (157) provided occupational and respiratory symptom information, pulmonary function measurements, and DNA for genotyping. The genotypes of 101 were TNF-308*1/*1, 47 were *1/*2, and nine were *2/*2. Current smokers whose genotype was *2/*2 or *1/*2 had lower values compared with other combinations of genotype and smoking status. Among *1/*1 homozygotes, current smokers had better percent of predicted forced expiratory volume in 1 second (P = 0.04) mean values than nonsmokers and better percent of predicted forced vital capacity than exsmokers (P = 0.017) or nonsmokers (P = 0.008). These results indicate the complexity of determining which workers will develop acute and chronic adverse pulmonary conditions in response to exposure to grain dust and the toxins in cigarette smoke interacting with their genotype.

Contractile-Ring Assembly in Fission Yeast Cytokinesis: Recent Advances and New Perspectives

PubMed Central

Lee, I-Ju; Coffman, Valerie C.; Wu, Jian-Qiu

2017-01-01

The fission yeast Schizosaccharomyces pombe is an excellent model organism to study cytokinesis. Here, we review recent advances on contractile-ring assembly in fission yeast. First, we summarize the assembly of cytokinesis nodes, the precursors of a normal contractile ring. IQGAP Rng2 and myosin essential light chain Cdc4 are recruited by the anillin-like protein Mid1, followed by the addition of other cytokinesis node proteins. Mid1 localization on the plasma membrane is stabilized by interphase node proteins. Second, we discuss proteins and processes that contribute to the search, capture, pull, and release mechanism of contractile-ring assembly. Actin filaments nucleated by formin Cdc12, the motor activity of myosin-II, the stiffness of the actin network, and severing of actin filaments by cofilin all play essential roles in contractile-ring assembly. Finally, we discuss the Mid1-independent pathway for ring assembly, and the possible mechanisms underlying the ring maturation and constriction. Collectively, we provide an overview of the current understanding of contractile-ring assembly and uncover future directions in studying cytokinesis in fission yeast. PMID:22887981

High-density lipoproteins protect endothelial cells from tumor necrosis factor-alpha-induced apoptosis.

PubMed

Sugano, M; Tsuchida, K; Makino, N

2000-06-16

High-density lipoproteins (HDL) levels have been shown to be inversely correlated with coronary heart disease, but the mechanisms of the direct protective effect of HDL on endothelial cells are not fully understood. The apoptosis of endothelial cells induced by cytokines and/or oxidized low-density lipoproteins, etc. may provide a mechanistic clue to the "response-to-injury" hypothesis of atherogenesis. Here we report that HDL prevent the apoptosis of human umbilical venous endothelial cells (HUVECs) induced by tumor necrosis factor-alpha (TNF-alpha) via an inhibition of CPP32-like protease activity. The incubation of HUVECs with TNF-alpha significantly increased the CPP32-like protease activity, and induced apoptosis. Preincubation of HUVECs with HDL before incubation with TNF-alpha significantly suppressed the increase in the CPP32-like protease activity, preventing apoptosis in a concentration-dependent manner. These results suggest that HDL prevent the suicide pathway leading to apoptosis of endothelial cells by decreasing the CPP32-like protease activity and that HDL thus play a protective role against the "response-to-injury" hypothesis of atherogenesis. Copyright 2000 Academic Press.

Treatment of rheumatoid arthritis with chimeric monoclonal antibodies to tumor necrosis factor alpha.

PubMed

Elliott, M J; Maini, R N; Feldmann, M; Long-Fox, A; Charles, P; Katsikis, P; Brennan, F M; Walker, J; Bijl, H; Ghrayeb, J

1993-12-01

To evaluate the safety and efficacy of a chimeric monoclonal antibody to tumor necrosis factor alpha (TNF alpha) in the treatment of patients with rheumatoid arthritis (RA). Twenty patients with active RA were treated with 20 mg/kg of anti-TNF alpha in an open phase I/II trial lasting 8 weeks. The treatment was well tolerated, with no serious adverse events. Significant improvements were seen in the Ritchie Articular Index, which fell from a median of 28 at study entry to a median of 6 by week 6 (P < 0.001), the swollen joint count, which fell from 18 to 5 (P < 0.001) over the same period, and in the other major clinical assessments. Serum C-reactive protein levels fell from a median of 39.5 mg/liter at study entry to 8 mg/liter at week 6 (P < 0.001), and significant decreases were also seen in serum amyloid A and interleukin-6 levels. Treatment with anti-TNF alpha was safe and well tolerated and resulted in significant clinical and laboratory improvements. These preliminary results support the hypothesis that TNF alpha is an important regulator in RA, and suggest that it may be a useful new therapeutic target in this disease.

SH2 domains of the p85 alpha subunit of phosphatidylinositol 3-kinase regulate binding to growth factor receptors.

PubMed Central

McGlade, C J; Ellis, C; Reedijk, M; Anderson, D; Mbamalu, G; Reith, A D; Panayotou, G; End, P; Bernstein, A; Kazlauskas, A

1992-01-01

The binding of cytoplasmic signaling proteins such as phospholipase C-gamma 1 and Ras GTPase-activating protein to autophosphorylated growth factor receptors is directed by their noncatalytic Src homology region 2 (SH2) domains. The p85 alpha regulatory subunit of phosphatidylinositol (PI) 3-kinase, which associates with several receptor protein-tyrosine kinases, also contains two SH2 domains. Both p85 alpha SH2 domains, when expressed individually as fusion proteins in bacteria, bound stably to the activated beta receptor for platelet-derived growth factor (PDGF). Complex formation required PDGF stimulation and was dependent on receptor tyrosine kinase activity. The bacterial p85 alpha SH2 domains recognized activated beta PDGF receptor which had been immobilized on a filter, indicating that SH2 domains contact autophosphorylated receptors directly. Several receptor tyrosine kinases within the PDGF receptor subfamily, including the colony-stimulating factor 1 receptor and the Steel factor receptor (Kit), also associate with PI 3-kinase in vivo. Bacterially expressed SH2 domains derived from the p85 alpha subunit of PI 3-kinase bound in vitro to the activated colony-stimulating factor 1 receptor and to Kit. We infer that the SH2 domains of p85 alpha bind to high-affinity sites on these receptors, whose creation is dependent on receptor autophosphorylation. The SH2 domains of p85 are therefore primarily responsible for the binding of PI 3-kinase to activated growth factor receptors. Images PMID:1372092

Serum concentrations of tumour necrosis factor-alpha (TNF-alpha) and soluble TNF-alpha receptor p55 in patients with hypothyroidism and hyperthyroidism before and after normalization of thyroid function.

PubMed

Díez, Juan J; Hernanz, Angel; Medina, Sonia; Bayón, Carmen; Iglesias, Pedro

2002-10-01

Tumour necrosis factor-alpha (TNF-alpha) is a cytokine with numerous immunological and metabolic activities. Receptors for TNF-alpha have been demonstrated in thyroid follicular cells and TNF-alpha and its receptors have been implicated in the cytotoxic mechanisms that characterize the thyroid destruction in autoimmune thyroid disease. In patients with Graves' disease, serum levels of TNF-alpha have been reported to be elevated and administration of TNF-alpha to humans has been shown to induce hormonal alterations resembling those seen in the nonthyroidal illness syndrome. To evaluate serum concentrations of TNF-alpha and the soluble receptor for TNF-alpha (sTNFR-I) in a group of patients with thyroid dysfunction before and after normalization of thyroid function with appropriate therapy. We studied 20 patients with hypothyroidism (18 women and 2 men, mean age +/- SD, 48.8 +/- 16.1 years) and 20 patients with hyperthyroidism (14 women and 6 men, age 44.6 +/- 15.9 years). Patients were assessed at the time of diagnosis and again after normalization of thyroid function tests with appropriate therapy. A group of 20 healthy subjects (15 women and 5 men, age 44.9 +/- 15.1 years) were also studied as a control group. All subjects were ambulatory and were studied as outpatients during visits to the endocrinology clinic. Serum concentrations of free T4 (FT4), total T3, TSH, TNF-alpha and sTNFR-I were measured in all subjects. TNF-alpha and sTNFR-I were measured using a quantitative enzyme immunoassay. In patients with hypothyroidism serum concentrations of TNF-alpha (3.17 +/- 1.18 pg/ml) and sTNFR-I (1273 +/- 364 pg/ml) were significantly higher than those found in controls (2.42 +/- 0.76 pg/ml, P < 0.05, and 971 +/- 235 pg/ml, P < 0.01, respectively). Normalization of thyroid function with l-thyroxine therapy did not significantly modify TNF-alpha or sTNFR-I levels. There were no differences in pre- and post-therapy values of TNF-alpha and sTNFR-I in patients with

Neocortical concentrations of neuropeptides in senile dementia of the Alzheimer and Lewy body type: comparison with Parkinson's disease and severity correlations.

PubMed

Leake, A; Perry, E K; Perry, R H; Jabeen, S; Fairbairn, A F; McKeith, I G; Ferrier, I N

1991-02-15

Corticotropin releasing hormone (CRH), somatostatin (SRIF), and arginine vasopressin (AVP) concentrations were estimated using radioimmunoassay in the temporal and occipital cortices in postmortem brain from patients clinically and neuropathologically diagnosed as senile dementia of the Lewy body type (SDLT), senile dementia of the Alzheimer type (SDAT), and Parkinson's disease (PD) and from neurologically normal controls. The concentration of temporal and occipital neocortical CRH was diminished in both SDAT and SDLT compared to control values, whereas SRIF was reduced only in temporal cortex in both these conditions. In contrast, the concentrations of both CRH and SRIF were unaltered in PD. The concentrations of AVP in SDLT, SDAT, and PD were similar to those found in the control groups. The decrement in SRIF, but not CRH, was found to be correlated with some indices of severity of illness in SDAT; a similar but nonsignificant trend for SRIF was observed in SDLT.

IgG1 antimycobacterial antibodies can reverse the inhibitory effect of pentoxifylline on tumour necrosis factor alpha (TNF-alpha) secreted by mycobacterial antigen-stimulated adherent cells.

PubMed

Thakurdas, S M; Hasan, Z; Hussain, R

2004-05-01

Chronic inflammation associated with cachexia, weight loss, fever and arthralgia is the hallmark of advanced mycobacterial diseases. These symptoms are attributed to the chronic stimulation of tumour necrosis factor (TNF)-alpha. Mycobacterial components directly stimulate adherent cells to secrete TNF-alpha. We have shown recently that IgG1 antimycobacterial antibodies play a role in augmenting TNF-alpha in purified protein derivative (PPD)-stimulated adherent cells from non-BCG-vaccinated donors. We now show that IgG1 antibodies can also augment TNF-alpha expression in stimulated adherent cells obtained from BCG-vaccinated donors and this augmentation is not linked to interleukin (IL)-10 secretion. In addition IgG1 antimycobacterial antibodies can reverse the effect of TNF-alpha blockers such as pentoxifylline and thalidomide. These studies therefore have clinical implications for anti-inflammatory drug treatments which are used increasingly to alleviate symptoms associated with chronic inflammation.

Gene polymorphisms of tumor necrosis factor alpha-308 and interleukin-10-1082 among asthmatic Egyptian children.

PubMed

Zedan, Magdy; Settin, Ahmed; Farag, Mohammad K; El-Bayoumi, Mohammed; El Regal, Mohammed Ezz; El Baz, Rizk; Osman, Engy

2008-01-01

Tumor necrosis factor (TNF) alpha-308 and interleukin (IL)-10(-1082) have potent inflammatory responses in the process of airway inflammation in asthma. The purpose of this study was to check for association of polymorphisms related to cytokine genes with susceptibility and severity of bronchial asthma in Egyptian children. Blood samples of 69 asthmatic children receiving treatment and follow-up at the Allergy and Respiratory Medicine Unit, Mansoura University Children Hospital, Mansoura, Egypt, were subjected to DNA extraction and amplification using polymerase chain reaction with sequence-specific primers for detection of single nucleotide polymorphisms in the promoter regions of cytokine genes TNF-alpha(-308(G-->A)), IL-10(-1082(G-->A)). Compared with normal controls, Egyptian asthmatic children showed a significant higher frequency of IL-10(-1082) G/G homozygosity genotype (p < 0.001; odds ratio [OR] = 7) with lower frequency of G/A heterozygosity genotype among cases. This finding also was detected in cases with persistent asthma and eczema. These cases showed significant lower frequency of TNF-alpha-308 G/A heterozygosity (p < 0.05; OR = 0.44). Also, male cases, cases with positive family history, and those patients with persistent types of asthma showed a higher frequency of TNF-alpha-308 G/G homozygosity. IL-10(-1082(G-->A)) G/G and TNF-alpha-308(G-->A) G/G may be a contributing factor in susceptibility as well as severity of asthma among Egyptian children. Separate studies should be specified relating these cytokine genotypes to response to various modalities in asthma therapy. This study reports that IL-10(-1082(G-->A)) G/G and TNF-alpha-308(G-->A) G/G genotypes may be contributing factors in susceptibility as well as in severity of asthma among Egyptian children. Separate studies may be specified relating these cytokine genotypes to response to various modalities in asthma therapy.

Method for high specific bioproductivity of .alpha.,.omega.-alkanedicarboxylic acids

DOEpatents

Mobley, David Paul; Shank, Gary Keith

2000-01-01

This invention provides a low-cost method of producing .alpha.,.omega.-alkanedicarboxylic acids. Particular bioconversion conditions result in highly efficient conversion of fatty acid, fatty acid ester, or alkane substrates to diacids. Candida tropicalis AR40 or similar yeast strains are grown in a medium containing a carbon source and a nitrogen source at a temperature of 31.degree. C. to 38.degree. C., while additional carbon source is continuously added, until maximum cell growth is attained. Within 0-3 hours of this point, substrate is added to the culture to initiate conversion. An .alpha.,.omega.-alkanedicarboxylic acid made according to this method is also provided.

EGF receptor tyrosine kinase inhibitors diminish transforming growth factor-alpha-induced pulmonary fibrosis.

PubMed

Hardie, William D; Davidson, Cynthia; Ikegami, Machiko; Leikauf, George D; Le Cras, Timothy D; Prestridge, Adrienne; Whitsett, Jeffrey A; Korfhagen, Thomas R

2008-06-01

Transforming growth factor-alpha (TGF-alpha) is a ligand for the EGF receptor (EGFR). EGFR activation is associated with fibroproliferative processes in human lung disease and animal models of pulmonary fibrosis. We determined the effects of EGFR tyrosine kinase inhibitors gefitinib (Iressa) and erlotinib (Tarceva) on the development and progression of TGF-alpha-induced pulmonary fibrosis. Using a doxycycline-regulatable transgenic mouse model of lung-specific TGF-alpha expression, we determined effects of treatment with gefitinib and erlotinib on changes in lung histology, total lung collagen, pulmonary mechanics, pulmonary hypertension, and expression of genes associated with synthesis of ECM and vascular remodeling. Induction in the lung of TGF-alpha caused progressive pulmonary fibrosis over an 8-wk period. Daily administration of gefitinib or erlotinib prevented development of fibrosis, reduced accumulation of total lung collagen, prevented weight loss, and prevented changes in pulmonary mechanics. Treatment of mice with gefitinib 4 wk after the induction of TGF-alpha prevented further increases in and partially reversed total collagen levels and changes in pulmonary mechanics and pulmonary hypertension. Increases in expression of genes associated with synthesis of ECM as well as decreases of genes associated with vascular remodeling were also prevented or partially reversed. Administration of gefitinib or erlotinib did not cause interstitial fibrosis or increases in lavage cell counts. Administration of small molecule EGFR tyrosine kinase inhibitors prevented further increases in and partially reversed pulmonary fibrosis induced directly by EGFR activation without inducing inflammatory cell influx or additional lung injury.

Inhibitory spectrum of alpha 2-plasmin inhibitor.

PubMed Central

Saito, H; Goldsmith, G H; Moroi, M; Aoki, N

1979-01-01

alpha 2-Plasmin inhibitor (alpha 2PI) has been recently characterized as a fast-reacting inhibitor of plasmin in human plasma and appears to play an important role in the regulation of fibrinolysis in vivo. We have studied the effect of purified alpha 2PI upon various proteases participating in human blood coagulation and kinin generation. At physiological concentration (50 microgram/ml), alpha 2PI inhibited the clot-promoting and prekallikrein-activating activity of Hageman factor fragments, the amidolytic, kininogenase, and clot-promoting activities of plasma kallikrein, and the clot-promoting properties of activated plasma thromboplastin antecedent (PTA, Factor XIa) and thrombin. alpha 2PI had minimal inhibitory effect on surface-bound activated PTA and activated Stuart factor (Factor Xa). alpha 2PI did not inhibit the activity of activated Christmas factor (Factor IXa) or urinary kallikrein. Heparin (1.5-2.0 units/ml) did not enhance the inhibitory function of alpha 2PI. These results suggest that, like other plasma protease inhibitors, alpha 2PI possesses a broad in vitro spectrum of inhibitory properties. PMID:156364

Inhibition of ribosome recruitment induces stress granule formation independently of eukaryotic initiation factor 2alpha phosphorylation.

PubMed

Mazroui, Rachid; Sukarieh, Rami; Bordeleau, Marie-Eve; Kaufman, Randal J; Northcote, Peter; Tanaka, Junichi; Gallouzi, Imed; Pelletier, Jerry

2006-10-01

Cytoplasmic aggregates known as stress granules (SGs) arise as a consequence of cellular stress and contain stalled translation preinitiation complexes. These foci are thought to serve as sites of mRNA storage or triage during the cell stress response. SG formation has been shown to require induction of eukaryotic initiation factor (eIF)2alpha phosphorylation. Herein, we investigate the potential role of other initiation factors in this process and demonstrate that interfering with eIF4A activity, an RNA helicase required for the ribosome recruitment phase of translation initiation, induces SG formation and that this event is not dependent on eIF2alpha phosphorylation. We also show that inhibition of eIF4A activity does not impair the ability of eIF2alpha to be phosphorylated under stress conditions. Furthermore, we observed SG assembly upon inhibition of cap-dependent translation after poliovirus infection. We propose that SG modeling can occur via both eIF2alpha phosphorylation-dependent and -independent pathways that target translation initiation.

Trimethyltin-activated cyclooxygenase stimulates tumor necrosis factor-alpha release from glial cells through reactive oxygen species.

PubMed

Viviani, B; Corsini, E; Pesenti, M; Galli, C L; Marinovich, M

2001-04-15

Exposure of a primary culture of glial cells to the classical neurotoxicant trimethyltin (TMT) results in the release of prostaglandin (PG)E(2) and tumor necrosis factor (TNF)-alpha. Prior treatment of glial cells with either the nonspecific inhibitor of cyclooxygenase and lypoxygenase eicosatetraynoic acid (ETYA) or the cyclooxygenase inhibitor indomethacin completely prevented TMT-induced PGE(2) production and TNF-alpha release, suggesting a role for cyclooxygenase metabolites in TMT-induced TNF-alpha release. Exposure of glial cells to increasing concentrations of PGE(2) or other prostanoids did not increase TNF-alpha synthesis, while the presence of exogenous PGE(2) during treatment of glial cells with TMT actually suppressed TNF-alpha release. The activation of arachidonic acid metabolism produces reactive oxygen species (ROS). Scavenging of ROS by means of the antioxidant trolox prevented the TMT-induced release of TNF-alpha from glial cells, while indomethacin was found to suppress ROS formation induced by 1 microM TMT in glial cells. These results suggest that activation of arachidonic acid metabolism causes TNF-alpha release through the production of ROS rather than PGE(2). Indeed, PGE(2) may exert negative feedback on the release of TNF-alpha. Copyright 2001 Academic Press.

Expression of tumour necrosis factor alpha and accumulation of fibronectin in coronary artery restenotic lesions retrieved by atherectomy.

PubMed Central

Clausell, N.; de Lima, V. C.; Molossi, S.; Liu, P.; Turley, E.; Gotlieb, A. I.; Adelman, A. G.; Rabinovitch, M.

1995-01-01

BACKGROUND--The formation of coronary artery neointima experimentally induced in piglets after cardiac transplantation is related to an immune-inflammatory reaction associated with increased expression of T cells and inflammatory mediators (tumour necrosis factor alpha and interleukin 1 beta) and upregulation of fibronectin. In vivo blockade of tumour necrosis factor alpha in rabbits after cardiac transplantation results in reduced neointimal formation. The objective of this study was to investigate the hypothesis that coronary restenosis after atherectomy or percutaneous balloon angioplasty is associated with a similar inflammatory cascade initiated by mechanical injury. METHODS--Specimens taken at coronary atherectomy were analysed from 16 patients. Nine had had the procedure performed twice, firstly, to remove a primary lesion, and secondly, to remove a restenotic lesion. Seven had percutaneous balloon angioplasty after removal of restenotic tissue. Coronary atherectomy specimens were analysed by immunohistochemistry for the presence of T cells, macrophages, major histocompatibility complex II, interleukin 1 beta, tumour necrosis factor alpha, fibronectin, and the receptor for hyaluronan mediated motility. RESULTS--The groups were clinically and angiographically similar with equivalent lumens before and after atherectomy. Restenotic lesions had increased expression of tumour necrosis factor alpha and fibronectin compared with the primary lesions (P < 0.05 for both). There was also a trend towards a greater number of T cells and increased expression of interleukin 1 beta. CONCLUSIONS--Restenosis is associated with increased expression of tumour necrosis factor alpha and fibronectin, suggesting that an immune-inflammatory reaction probably contributes to neointimal formation and may represent a form of wound healing and repair secondary to mechanical injury. Images PMID:7626352

Etanercept prevents decrease of cochlear blood flow dose-dependently caused by tumor necrosis factor alpha.

PubMed

Ihler, Friedrich; Sharaf, Kariem; Bertlich, Mattis; Strieth, Sebastian; Reichel, Christoph A; Berghaus, Alexander; Canis, Martin

2013-07-01

Tumor necrosis factor alpha (TNF-alpha) is a mediator of inflammation and microcirculation in the cochlea. This study aimed to quantify the effect of a local increase of TNF-alpha and study the effect of its interaction with etanercept on cochlear microcirculation. Cochlear lateral wall vessels were exposed surgically and assessed by intravital microscopy in guinea pigs in vivo. First, 24 animals were randomly distributed into 4 groups of 6 each. Exposed vessels were superfused repeatedly either with 1 of 3 different concentrations of TNF-alpha (5.0, 0.5, and 0.05 ng/mL) or with placebo (0.9% saline solution). Second, 12 animals were randomly distributed into 2 groups of 6 each. Vessels were pretreated with etanercept (1.0 microg/ mL) or placebo (0.9% saline solution), and then treated by repeated superfusion with TNF-alpha (5.0 ng/mL). TNF-alpha was shown to be effective in decreasing cochlear blood flow at a dose of 5.0 ng/mL (p < 0.01, analysis of variance on ranks). Lower concentrations or placebo treatment did not lead to significant changes. After pretreatment with etanercept, TNF-alpha at a dose of 5.0 ng/mL no longer led to a change in cochlear blood flow. The decreasing effect that TNF-alpha has on cochlear blood flow is dose-dependent. Etanercept abrogates this effect.

Contributions of toluene and alpha-pinene to SOA formed in an irradiated toluene/alpha-pinene/NO(x)/ air mixture: comparison of results using 14C content and SOA organic tracer methods.

PubMed

Offenberg, John H; Lewis, Charles W; Lewandowski, Michael; Jaoui, Mohammed; Kleindienst, Tadeusz E; Edney, Edward O

2007-06-01

An organic tracer method, recently proposed for estimating individual contributions of toluene and alpha-pinene to secondary organic aerosol (SOA) formation, was evaluated by conducting a laboratory study where a binary hydrocarbon mixture, containing the anthropogenic aromatic hydrocarbon, toluene, and the biogenic monoterpene, alpha-pinene, was irradiated in air in the presence of NO(x) to form SOA. The contributions of toluene and alpha-pinene to the total SOA concentration, calculated using the organic tracer method, were compared with those obtained with a more direct 14C content method. In the study, SOA to SOC ratios of 2.07 +/- 0.08 and 1.41 +/- 0.04 were measured for toluene and (alpha-pinene SOA, respectively. The individual tracer-based SOA contributions of 156 microg m(-3) for toluene and 198 microg m(-)3 for alpha-pinene, which together accounted for 82% of the gravimetrically determined total SOA concentration, compared well with the 14C values of 182 and 230 microg m(-3) measured for the respective SOA precursors. While there are uncertainties associated with the organic tracer method, largely due to the chemical complexity of SOA forming chemical mechanisms, the results of this study suggest the organic tracer method may serve as a useful tool for determining whether a precursor hydrocarbon is a major SOA contributor.

Confirmatory Factor Analysis of the Trinity Inventory of Precursors to Suicide (TIPS) and Its Relationship to Hopelessness and Depression

ERIC Educational Resources Information Center

Smyth, Caroline L.; MacLachlan, Malcolm

2005-01-01

Numerous existing measures assess attitudes toward suicide yet fail to account for contextual factors. The Trinity Inventory of Precursors to Suicide (TIPS) is presented as an alternative, with implications for the development of prevention programs. Having previously reported exploratory analysis of the TIPS; confirmatory factor analysis and…

Agrobacterium tumefaciens-mediated transformation of oleaginous yeast Lipomyces species.

PubMed

Dai, Ziyu; Deng, Shuang; Culley, David E; Bruno, Kenneth S; Magnuson, Jon K

2017-08-01

Interest in using renewable sources of carbon, especially lignocellulosic biomass, for the production of hydrocarbon fuels and chemicals has fueled interest in exploring various organisms capable of producing hydrocarbon biofuels and chemicals or their precursors. The oleaginous (oil-producing) yeast Lipomyces starkeyi is the subject of active research regarding the production of triacylglycerides as hydrocarbon fuel precursors using a variety of carbohydrate and nutrient sources. The genome of L. starkeyi has been published, which opens the door to production strain improvements through the development and use of the tools of synthetic biology for this oleaginous species. The first step in establishment of synthetic biology tools for an organism is the development of effective and reliable transformation methods with suitable selectable marker genes and demonstration of the utility of the genetic elements needed for expression of introduced genes or deletion of endogenous genes. Chemical-based methods of transformation have been published but suffer from low efficiency. To address these problems, Agrobacterium-mediated transformation was investigated as an alternative method for L. starkeyi and other Lipomyces species. In this study, Agrobacterium-mediated transformation was demonstrated to be effective in the transformation of both L. starkeyi and other Lipomyces species. The deletion of the peroxisomal biogenesis factor 10 gene was also demonstrated in L. starkeyi. In addition to the bacterial antibiotic selection marker gene hygromycin B phosphotransferase, the bacterial β-glucuronidase reporter gene under the control of L. starkeyi translation elongation factor 1α promoter was also stably expressed in six different Lipomyces species. The results from this study demonstrate that Agrobacterium-mediated transformation is a reliable and effective genetic tool for homologous recombination and expression of heterologous genes in L. starkeyi and other Lipomyces

Tumour necrosis factor alpha changes porcine intestinal ion transport through a paracrine mechanism involving prostaglandins.

PubMed Central

Kandil, H M; Berschneider, H M; Argenzio, R A

1994-01-01

Prostaglandins stimulate electrogenic anion secretion and inhibit sodium chloride absorption in cryptosporidium induced pig diarrhoea. Because tumour necrosis factor alpha (TNF alpha) is an early mediator of inflammation and stimulates prostaglandin secretion, we investigated its effect on intestinal ion transport. Cryptosporidium infected pig ileum showed higher macrophage infiltration and tissue TNF alpha-like activity than uninfected tissues (p < 0.05, n = 4 and p < 0.05, n = 12, respectively). TNF alpha treatment of control porcine ileal mucosa increased the short circuit current (Isc), a measurement of net anion secretion in this model (p < 0.001, n = 23). This effect was blocked by 10(-6) M indomethacin and Cl- replacement. Neither acute treatment nor preincubation of colonic intestinal epithelial cell monolayers (T84) with TNF alpha stimulated the Isc. However, co-mounting of TNF alpha preincubated pig jejunal fibroblasts (P2JF) monolayers back to back with untreated T84 monolayers dose-dependently induced an indomethacin sensitive increase in Isc compared with values in untreated co-mounted monolayers (p < 0.001, n = 11). These data suggest that in infectious diarrhoea, TNF alpha may induce Cl- secretion through a paracrine mechanism involving prostaglandin release from subepithelial cells, for example fibroblasts. PMID:8063221

Radiocurability by Targeting Tumor Necrosis Factor-{alpha} Using a Bispecific Antibody in Carcinoembryonic Antigen Transgenic Mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Larbouret, Christel; Robert, Bruno; Linard, Christine

2007-11-15

Purpose: Tumor necrosis factor-{alpha} (TNF-{alpha}) enhances radiotherapy (RT) killing of tumor cells in vitro and in vivo. To overcome systemic side effects, we used a bispecific antibody (BsAb) directed against carcinoembryonic antigen (CEA) and TNF-{alpha} to target this cytokine in a CEA-expressing colon carcinoma. We report the evaluation of this strategy in immunocompetent CEA-transgenic mice. Methods and Materials: The murine CEA-transfected colon carcinoma MC-38 was used for all experiments. In vitro, clonogenic assays were performed after RT alone, TNF-{alpha} alone, and RT plus TNF-{alpha}. In vivo, the mice were randomly assigned to treatment groups: control, TNF-{alpha}, BsAb, BsAb plus TNF-{alpha},more » RT, RT plus TNF-{alpha}, and RT plus BsAb plus TNF-{alpha}. Measurements of endogenous TNF-{alpha} mRNA levels and evaluation of necrosis (histologic evaluation) were assessed per treatment group. Results: In vitro, combined RT plus TNF-{alpha} resulted in a significant decrease in the survival fraction at 2 Gy compared with RT alone (p < 0.00001). In vivo, we observed a complete response in 5 (50%) of 10, 2 (20%) of 10, 2 (18.2%) of 11, and 0 (0%) of 12 treated mice in the RT plus BsAb plus TNF-{alpha}, RT plus TNF-{alpha}, RT alone, and control groups, respectively. This difference was statistically significant when TNF-{alpha} was targeted with the BsAb (p = 0.03). The addition of exogenous TNF-{alpha} to RT significantly increased the endogenous TNF-{alpha} mRNA level, particularly when TNF-{alpha} was targeted with BsAb (p < 0.01). The percentages of necrotic area were significantly augmented in the RT plus BsAb plus TNF-{alpha} group. Conclusion: These results suggest that targeting TNF-{alpha} with the BsAb provokes RT curability in a CEA-expressing digestive tumor syngenic model and could be considered as a solid rationale for clinical trials.« less

Transcription factor genes essential for cell proliferation and replicative lifespan in budding yeast

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kamei, Yuka; Tai, Akiko; Dakeyama, Shota

Many of the lifespan-related genes have been identified in eukaryotes ranging from the yeast to human. However, there is limited information available on the longevity genes that are essential for cell proliferation. Here, we investigated whether the essential genes encoding DNA-binding transcription factors modulated the replicative lifespan of Saccharomyces cerevisiae. Heterozygous diploid knockout strains for FHL1, RAP1, REB1, and MCM1 genes showed significantly short lifespan. {sup 1}H-nuclear magnetic resonance analysis indicated a characteristic metabolic profile in the Δfhl1/FHL1 mutant. These results strongly suggest that FHL1 regulates the transcription of lifespan related metabolic genes. Thus, heterozygous knockout strains could be themore » potential materials for discovering further novel lifespan genes. - Highlights: • Involvement of yeast TF genes essential for cell growth in lifespan was evaluated. • The essential TF genes, FHL1, RAP1, REB1, and MCM1, regulate replicative lifespan. • Heterozygous deletion of FHL1 changes cellular metabolism related to lifespan.« less

Involvement of Mst1 in tumor necrosis factor-{alpha}-induced apoptosis of endothelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ohtsubo, Hideki; Ichiki, Toshihiro; Imayama, Ikuyo

2008-03-07

Mammalian sterile 20-kinase 1 (Mst1), a member of the sterile-20 family protein kinase, plays an important role in the induction of apoptosis. However, little is know about the physiological activator of Mst1 and the role of Mst1 in endothelial cells (ECs). We examined whether Mst1 is involved in the tumor necrosis factor (TNF)-{alpha}-induced apoptosis of ECs. Western blot analysis revealed that TNF-{alpha} induced activation of caspase 3 and Mst1 in a time- and dose-dependent manner. TNF-{alpha}-induced Mst1 activation is almost completely prevented by pretreatment with Z-DEVD-FMK, a caspase 3 inhibitor. Nuclear staining with Hoechst 33258 and fluorescence-activated cell sorting ofmore » propidium iodide-stained cells showed that TNF-{alpha} induced apoptosis of EC. Diphenyleneiodonium, an inhibitor of NADPH oxidase, and N-acetylcysteine, a potent antioxidant, also inhibited TNF-{alpha}-induced activation of Mst1 and caspase 3, as well as apoptosis. Knockdown of Mst1 expression by short interfering RNA attenuated TNF-{alpha}-induced apoptosis but not cleavage of caspase 3. These results suggest that Mst1 plays an important role in the induction of TNF-{alpha}-induced apoptosis of EC. However, positive feedback mechanism between Mst1 and caspase 3, which was shown in the previous studies, was not observed. Inhibition of Mst1 function may be beneficial for maintaining the endothelial integrity and inhibition of atherogenesis.« less

Oleaginous yeasts: Promising platforms for the production of oleochemicals and biofuels.

PubMed

Adrio, José L

2017-09-01

Oleaginous yeasts have a unique physiology that makes them the best suited hosts for the production of lipids, oleochemicals, and diesel-like fuels. Their high lipogenesis, capability of growing on many different carbon sources (including lignocellulosic sugars), easy large-scale cultivation, and an increasing number of genetic tools are some of the advantages that have encouraged their use to develop sustainable processes. This mini-review summarizes the metabolic engineering strategies developed in oleaginous yeasts within the last 2 years to improve process metrics (titer, yield, and productivity) for the production of lipids, free fatty acids, fatty acid-based chemicals (e.g., fatty alcohols, fatty acid ethyl esters), and alkanes. During this short period of time, tremendous progress has been made in Yarrowia lipolytica, the model oleaginous yeast, which has been engineered to improve lipid production by different strategies including increasing lipogenic pathway flux and biosynthetic precursors, and blocking degradation pathways. Moreover, remarkable advances have also been reported in Rhodosporidium toruloides and Lipomyces starkey despite the limited genetic tools available for these two very promising hosts. Biotechnol. Bioeng. 2017;114: 1915-1920. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

The yeast Hot1 transcription factor is critical for activating a single target gene, STL1

PubMed Central

Bai, Chen; Tesker, Masha; Engelberg, David

2015-01-01

Transcription factors are commonly activated by signal transduction cascades and induce expression of many genes. They therefore play critical roles in determining the cell's fate. The yeast Hog1 MAP kinase pathway is believed to control the transcription of hundreds of genes via several transcription factors. To identify the bona fide target genes of Hog1, we inducibly expressed the spontaneously active variant Hog1D170A+F318L in cells lacking the Hog1 activator Pbs2. This system allowed monitoring the effects of Hog1 by itself. Expression of Hog1D170A+F318L in pbs2∆ cells imposed induction of just 105 and suppression of only 26 transcripts by at least twofold. We looked for the Hog1-responsive element within the promoter of the most highly induced gene, STL1 (88-fold). A novel Hog1 responsive element (HoRE) was identified and shown to be the direct target of the transcription factor Hot1. Unexpectedly, we could not find this HoRE in any other yeast promoter. In addition, the only gene whose expression was abolished in hot1∆ cells was STL1. Thus Hot1 is essential for transcription of just one gene, STL1. Hot1 may represent a class of transcription factors that are essential for transcription of a very few genes or even just one. PMID:25904326

Yeast Los1p Has Properties of an Exportin-Like Nucleocytoplasmic Transport Factor for tRNA

PubMed Central

Hellmuth, Klaus; Lau, Denise M.; Bischoff, F. Ralf; Künzler, Markus; Hurt, Ed; Simos, George

1998-01-01

Saccharomyces cerevisiae Los1p, which is genetically linked to the nuclear pore protein Nsp1p and several tRNA biogenesis factors, was recently grouped into the family of importin/karyopherin-β-like proteins on the basis of its sequence similarity. In a two-hybrid screen, we identified Nup2p as a nucleoporin interacting with Los1p. Subsequent purification of Los1p from yeast demonstrates its physical association not only with Nup2p but also with Nsp1p. By the use of the Gsp1p-G21V mutant, Los1p was shown to preferentially bind to the GTP-bound form of yeast Ran. Furthermore, overexpression of full-length or N-terminally truncated Los1p was shown to have dominant-negative effects on cell growth and different nuclear export pathways. Finally, Los1p could interact with Gsp1p-GTP, but only in the presence of tRNA, as revealed in an indirect in vitro binding assay. These data confirm the homology between Los1p and the recently identified human exportin for tRNA and reinforce the possibility of a role for Los1p in nuclear export of tRNA in yeast. PMID:9774653

Treatment of recalcitrant pemphigus vulgaris with the tumor necrosis factor alpha antagonist etanercept.

PubMed

Berookhim, Boback; Fischer, Harry D; Weinberg, Jeffrey M

2004-10-01

The treatment of pemphigus vulgaris (PV) is generally regarded as challenging. Patients with the disease require long-term systemic therapy, creating concern for the toxicities of these treatments. Corticosteroids, as drugs of first choice, often must be combined with steroid-sparing agents to prevent hazardous long-term side effects. We describe a 62-year-old woman with long-standing PV whose cutaneous disease responded to therapy with the tumor necrosis factor alpha (TNF-alpha) antagonist etanercept, which was started for treatment of her inflammatory seronegative arthritis. To our knowledge, this is the first report of its efficacy in the treatment of PV.

Generation of tumour-necrosis-factor-alpha-specific affibody molecules capable of blocking receptor binding in vitro.

PubMed

Jonsson, Andreas; Wållberg, Helena; Herne, Nina; Ståhl, Stefan; Frejd, Fredrik Y

2009-08-17

Affibody molecules specific for human TNF-alpha (tumour necrosis factor-alpha) were selected by phage-display technology from a library based on the 58-residue Protein A-derived Z domain. TNF-alpha is a proinflammatory cytokine involved in several inflammatory diseases and, to this day, four TNF-alpha-blocking protein pharmaceuticals have been approved for clinical use. The phage selection generated 18 unique cysteine-free affibody sequences of which 12 were chosen, after sequence cluster analysis, for characterization as proteins. Biosensor binding studies of the 12 Escherichia coli-produced and IMAC (immobilized-metal-ion affinity chromatography)-purified affibody molecules revealed three variants that demonstrated the strongest binding to human TNF-alpha. These three affibody molecules were subjected to kinetic binding analysis and also tested for their binding to mouse, rat and pig TNF-alpha. For ZTNF-alpha:185, subnanomolar affinity (KD=0.1-0.5 nM) for human TNF-alpha was demonstrated, as well as significant binding to TNF-alpha from the other species. Furthermore, the binding site was found to overlap with the binding site for the TNF-alpha receptor, since this interaction could be efficiently blocked by the ZTNF-alpha:185 affibody. When investigating six dimeric affibody constructs with different linker lengths, and one trimeric construct, it was found that the inhibition of the TNF-alpha binding to its receptor could be further improved by using dimers with extended linkers and/or a trimeric affibody construct. The potential implication of the results for the future design of affibody-based reagents for the diagnosis of inflammation is discussed.

Shikonins, phytocompounds from Lithospermum erythrorhizon, inhibit the transcriptional activation of human tumor necrosis factor alpha promoter in vivo.

PubMed

Staniforth, Vanisree; Wang, Sheng-Yang; Shyur, Lie-Fen; Yang, Ning-Sun

2004-02-13

Tumor necrosis factor alpha (TNF-alpha) contributes to the pathogenesis of both acute and chronic inflammatory diseases and has been a target for the development of new anti-inflammatory drugs. Shikonins, the naphthoquinone pigments present in the root tissues of Lithospermum erythrorhizon Sieb. et Zucc. (Boraginaceae), have been reported to exert anti-inflammatory effects both in vitro and in vivo. In this study, we evaluated the effects of shikonin and its derivatives on the transcriptional activation of human TNF-alpha promoter in a gene gun-transfected mouse skin system by using a luciferase reporter gene assay. The crude plant extract of L. erythrorhizon as well as derived individual compounds shikonin, isobutyryl shikonin, acetyl shikonin, dimethylacryl shikonin and isovaleryl shikonin showed significant dose-dependent inhibition of TNF-alpha promoter activation. Among the tested compounds, shikonin and isobutyryl shikonin exhibited the highest inhibition of TNF-alpha promoter activation and also showed significant suppression of transgenic human TNF-alpha mRNA expression and protein production. We demonstrated that shikonin-inhibitory response was retained in the core TNF-alpha promoter region containing the TATA box and a 48-bp downstream sequence relative to the transcription start site. Further our results indicated that shikonin suppressed the basal transcription and activator-regulated transcription of TNF-alpha by inhibiting the binding of transcription factor IID protein complex (TATA box-binding protein) to TATA box. These in vivo results suggest that shikonins inhibit the transcriptional activation of the human TNF-alpha promoter through interference with the basal transcription machinery. Thus, shikonins may have clinical potential as anti-inflammatory therapeutics.

The construction of recombinant industrial yeasts free of bacterial sequences by directed gene replacement into a nonessential region of the genome.

PubMed

Xiao, W; Rank, G H

1989-03-15

The yeast SMR1 gene was used as a dominant resistance-selectable marker for industrial yeast transformation and for targeting integration of an economically important gene at the homologous ILV2 locus. A MEL1 gene, which codes for alpha-galactosidase, was inserted into a dispensable upstream region of SMR1 in vitro; different treatments of the plasmid (pWX813) prior to transformation resulted in 3' end, 5' end and replacement integrations that exhibited distinct integrant structures. One-step replacement within a nonessential region of the host genome generated a stable integration of MEL1 devoid of bacterial plasmid DNA. Using this method, we have constructed several alpha-galactosidase positive industrial Saccharomyces strains. Our study provides a general method for stable gene transfer in most industrial Saccharomyces yeasts, including those used in the baking, brewing (ale and lager), distilling, wine and sake industries, with solely nucleotide sequences of interest. The absence of bacterial DNA in the integrant structure facilitates the commercial application of recombinant DNA technology in the food and beverage industry.

Nonneutral plasma diagnostic commissioning for the ALPHA Antihydrogen experiment

NASA Astrophysics Data System (ADS)

Konewko, S.; Friesen, T.; Tharp, T. D.; Alpha Collaboration

2017-10-01

The ALPHA experiment at CERN creates antihydrogen by mixing antiproton and positron plasmas. Diagnostic measurements of the precursor plasmas are performed using a diagnostic suite, colloquially known as the ``stick.'' This stick has a variety of sensors and is able to move to various heights to align the desired diagnostic with the beamline. A cylindrical electrode, a faraday cup, an electron gun, and a microchannel-plate detector (MCP) are regularly used to control and diagnose plasmas in ALPHA. We have designed, built, and tested a new, upgraded stick which includes measurement capabilities in both beamline directions.

Yeast ratio is a critical factor for sequential fermentation of papaya wine by Williopsis saturnus and Saccharomyces cerevisiae

PubMed Central

Lee, Pin-Rou; Kho, Stephanie Hui Chern; Yu, Bin; Curran, Philip; Liu, Shao-Quan

2013-01-01

Summary The growth kinetics and fermentation performance of Williopsis saturnus and Saccharomyces cerevisiae at ratios of 10:1, 1:1 and 1:10 (W.:S.) were studied in papaya juice with initial 7-day fermentation by W. saturnus, followed by S. cerevisiae. The growth kinetics of W. saturnus were similar at all ratios, but its maximum cell count decreased as the proportion of S. cerevisiae was increased. Conversely, there was an early death of S. cerevisiae at the ratio of 10:1. Williopsis saturnus was the dominant yeast at 10:1 ratio that produced papaya wine with elevated concentrations of acetate esters. On the other hand, 1:1 and 1:10 ratios allowed the coexistence of both yeasts which enabled the flavour-enhancing potential of W. saturnus as well as the ethyl ester and alcohol-producing abilities of S. cerevisiae. In particular, 1:1 and 1:10 ratios resulted in production of more ethyl esters, alcohols and 2-phenylethyl acetate. However, the persistence of both yeasts at 1:1 and 1:10 ratios led to formation of high levels of acetic acid. The findings suggest that yeast ratio is a critical factor for sequential fermentation of papaya wine by W. saturnus and S. cerevisiae as a strategy to modulate papaya wine flavour. PMID:23171032

Yeast ratio is a critical factor for sequential fermentation of papaya wine by Williopsis saturnus and Saccharomyces cerevisiae.

PubMed

Lee, Pin-Rou; Kho, Stephanie Hui Chern; Yu, Bin; Curran, Philip; Liu, Shao-Quan

2013-07-01

The growth kinetics and fermentation performance of Williopsis saturnus and Saccharomyces cerevisiae at ratios of 10:1, 1:1 and 1:10 (W.:S.) were studied in papaya juice with initial 7-day fermentation by W.saturnus, followed by S. cerevisiae. The growth kinetics of W. saturnus were similar at all ratios, but its maximum cell count decreased as the proportion of S. cerevisiae was increased. Conversely, there was an early death of S. cerevisiae at the ratio of 10:1. Williopsis saturnus was the dominant yeast at 10:1 ratio that produced papaya wine with elevated concentrations of acetate esters. On the other hand, 1:1 and 1:10 ratios allowed the coexistence of both yeasts which enabled the flavour-enhancing potential of W.saturnus as well as the ethyl ester and alcohol-producing abilities of S. cerevisiae. In particular, 1:1 and 1:10 ratios resulted in production of more ethyl esters, alcohols and 2-phenylethyl acetate. However, the persistence of both yeasts at 1:1 and 1:10 ratios led to formation of high levels of acetic acid. The findings suggest that yeast ratio is a critical factor for sequential fermentation of papaya wine by W.saturnus and S. cerevisiae as a strategy to modulate papaya wine flavour. © 2012 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

Yeast Based Sensors

NASA Astrophysics Data System (ADS)

Shimomura-Shimizu, Mifumi; Karube, Isao

Since the first microbial cell sensor was studied by Karube et al. in 1977, many types of yeast based sensors have been developed as analytical tools. Yeasts are known as facultative anaerobes. Facultative anaerobes can survive in both aerobic and anaerobic conditions. The yeast based sensor consisted of a DO electrode and an immobilized omnivorous yeast. In yeast based sensor development, many kinds of yeast have been employed by applying their characteristics to adapt to the analyte. For example, Trichosporon cutaneum was used to estimate organic pollution in industrial wastewater. Yeast based sensors are suitable for online control of biochemical processes and for environmental monitoring. In this review, principles and applications of yeast based sensors are summarized.

Genetic manipulation of murine embryonic stem cells with enhanced green fluorescence protein and sulfatase-modifying factor I genes.

PubMed

Zhao, Guoying; Karageorgos, Litsa; Hutchinson, Rhonda G; Hopwood, John J; Hemsley, Kim

2010-05-01

Mucopolysaccharidosis type IIIA (MPS IIIA) is a lysosomal storage disorder (LSD) in which an absence of sulfamidase results in incomplete degradation and subsequent accumulation of its substrate, heparan sulfate. Most neurodegenerative LSD remain untreatable. However, therapy options, such as gene, enzyme end cell therapy, are under investigation. Previously, we have constructed an embryonic stem (ES) cell line (NS21) that over-expresses human sulphamidase as a potential treatment for murine MPS IIIA. In the present study the sulfatase-modifying factor I (SUMF1) and enhanced green fluorescence protein (eGFP) genes were co-introduced under a cytomegalovirus (CMV) promoter into NS21 cells, to enhance further sulfamidase activity and provide a marker for in vivo cell tracking, respectively. eGFP was also introduced under the control of the human elongation factor-1alpha (hEF-1alpha) promoter to compare the stability of transgene expression. During differentiation of ES cells into glial precursors, SUMF1 was down-regulated and was hardly detectable by day 18 of differentiation. Likewise, eGFP expression was heterogeneous and highly unstable. Use of a human EF-1alpha promoter resulted in more homogeneous eGFP expression, with approximately 50% of cells eGFP positive following differentiation into glial precursors. Compared with NS21 cells, the outgrowth of eGFP-expressing cells was not as confluent when differentiated into glial precursors. Our data suggest that SUMF1 enhances sulfamidase activity in ES cells, hEF-1alpha is a stronger promoter than CMV for ES cells and over-expression of eGFP may affect cell growth and contribute to unstable gene expression.

Temporary reversal by topotecan of marked insulin resistance in a patient with myelodysplastic syndrome: case report and possible mechanism for tumor necrosis factor alpha (TNF-alpha)-induced insulin resistance.

PubMed

Huntington, M O; Krell, K E; Armour , W E; Liljenquist, J E

2001-06-01

Tumor necrosis factor-alpha (TNF-alpha) is an important mediator of insulin resistance in obesity and diabetes through its ability to decrease the tyrosine kinase activity of the insulin receptor. We report here a remarkable degree of insulin resistance in a patient with adult respiratory distress syndrome and myelodysplasia.

Purification and characterization of moschins, arginine-glutamate-rich proteins with translation-inhibiting activity from brown pumpkin (Cucurbita moschata) seeds.

PubMed

Ng, T B; Parkash, A; Tso, W W

2002-10-01

From fresh brown pumpkin seeds, two proteins with a molecular mass of 12kDa and an N-terminal sequence rich in arginine and glutamate residues were obtained. The protein designated alpha-moschin closely resembled the fruitfly programmed-cell death gene product and the protein designated beta-moschin demonstrated striking similarity to prepro 2S albumin in N-terminal sequence. alpha- and beta-moschins inhibited translation in the rabbit reticulocyte lysate system with an IC(50) of 17 microM and 300nM, respectively.

Risk of Lymphoma in Patients With Inflammatory Bowel Disease Treated With Anti-Tumor Necrosis Factor Alpha Agents: A Systematic Review and Meta-analysis.

PubMed

Yang, Chen; Huang, Junlin; Huang, Xiaowen; Huang, Shaozhuo; Cheng, Jiaxin; Liao, Weixin; Chen, Xuewen; Wang, Xueyi; Dai, Shixue

2018-05-12

The association between anti-tumor necrosis factor alpha agents and the risk of lymphoma in patients with inflammatory bowel disease has already been sufficiently reported. However, the results of these studies are inconsistent. Hence, this analysis was conducted to investigate whether anti-tumor necrosis factor alpha agents can increase the risk of lymphoma in inflammatory bowel disease patients. MEDLINE, EMBASE and the Cochrane Library were searched to identify relevant studies which evaluated the risk of lymphoma in inflammatory bowel disease patients treated with anti-tumor necrosis factor alpha agents. A random-effects meta-analysis was performed to calculate the pooled incidence rate ratios as well as risk ratios. Twelve studies comprising 285811 participants were included. The result showed that there was no significantly increased risk of lymphoma between anti-tumor necrosis factor alpha agents exposed and anti-tumor necrosis factor alpha agents unexposed groups (random effects: incidence rate ratio [IRR], 1.43 95%CI, 0.91-2.25, p= 0.116; random effects: risk ratio [RR], 0.83 95%CI, 0.47-1.48, p=0.534). However, monotherapy of anti-tumor necrosis factor alpha agents (random effects: IRR=1.65, 95%CI, 1.16-2.35; p=0.006; random effects: RR=1.00, 95%CI, 0.39-2.59; p=0.996) or combination therapy (random effects: IRR=3.36, 95%CI, 2.23-5.05; p< 0.001; random effects: RR=1.90, 95%CI, 0.66-5.44; p=0.233) can significantly increase the risk of lymphoma. Exposition of anti-tumor necrosis factor alpha agents in patients with inflammatory bowel disease is not associated with a higher risk of lymphoma. Combination therapy and anti-tumor necrosis factor alpha agents monotherapy can significantly increase the risk of lymphoma in patients with inflammatory bowel disease.

Improved fermentation performance of a lager yeast after repair of its AGT1 maltose and maltotriose transporter genes.

PubMed

Vidgren, Virve; Huuskonen, Anne; Virtanen, Hannele; Ruohonen, Laura; Londesborough, John

2009-04-01

The use of more concentrated, so-called high-gravity and very-high-gravity (VHG) brewer's worts for the manufacture of beer has economic and environmental advantages. However, many current strains of brewer's yeasts ferment VHG worts slowly and incompletely, leaving undesirably large amounts of maltose and especially maltotriose in the final beers. alpha-Glucosides are transported into Saccharomyces yeasts by several transporters, including Agt1, which is a good carrier of both maltose and maltotriose. The AGT1 genes of brewer's ale yeast strains encode functional transporters, but the AGT1 genes of the lager strains studied contain a premature stop codon and do not encode functional transporters. In the present work, one or more copies of the AGT1 gene of a lager strain were repaired with DNA sequence from an ale strain and put under the control of a constitutive promoter. Compared to the untransformed strain, the transformants with repaired AGT1 had higher maltose transport activity, especially after growth on glucose (which represses endogenous alpha-glucoside transporter genes) and higher ratios of maltotriose transport activity to maltose transport activity. They fermented VHG (24 degrees Plato) wort faster and more completely, producing beers containing more ethanol and less residual maltose and maltotriose. The growth and sedimentation behaviors of the transformants were similar to those of the untransformed strain, as were the profiles of yeast-derived volatile aroma compounds in the beers.

Post-fermentative production of glutathione by baker's yeast (S. cerevisiae) in compressed and dried forms.

PubMed

Musatti, Alida; Manzoni, Matilde; Rollini, Manuela

2013-01-25

The study was aimed at investigating the best biotransformation conditions to increase intracellular glutathione (GSH) levels in samples of baker's yeast (Saccharomyces cerevisiae) employing either the commercially available compressed and dried forms. Glucose, GSH precursors amino acids, as well as other cofactors, were dissolved in a biotransformation solution and yeast cells were added (5%dcw). Two response surface central composite designs (RSCCDs) were performed in sequence: in the first step the influence of amino acid composition (cysteine, glycine, glutamic acid and serine) on GSH accumulation was investigated; once their formulation was set up, the influence of other components was studied. Initial GSH content was found 0.53 and 0.47%dcw for compressed and dried forms. GSH accumulation ability of baker's yeast in compressed form was higher at the beginning of shelf life, that is, in the first week, and a maximum of 2.04%dcw was obtained. Performance of yeast in dried form was not found satisfactory, as the maximum GSH level was 1.18%dcw. When cysteine lacks from the reaction solution, yeast cells do not accumulate GSH. With dried yeast, the highest GSH yields occurred when cysteine was set at 3 g/L, glycine and glutamic acid at least at 4 g/L, without serine. Employing compressed yeast, the highest GSH yields occurred when cysteine and glutamic acid were set at 2-3 g/L, while glycine and serine higher than 2 g/L. Results allowed to set up an optimal and feasible procedure to obtain GSH-enriched yeast biomass, with up to threefold increase with respect to initial content. Copyright © 2012 Elsevier B.V. All rights reserved.

Chromatin and Transcription in Yeast

PubMed Central

Rando, Oliver J.; Winston, Fred

2012-01-01

Understanding the mechanisms by which chromatin structure controls eukaryotic transcription has been an intense area of investigation for the past 25 years. Many of the key discoveries that created the foundation for this field came from studies of Saccharomyces cerevisiae, including the discovery of the role of chromatin in transcriptional silencing, as well as the discovery of chromatin-remodeling factors and histone modification activities. Since that time, studies in yeast have continued to contribute in leading ways. This review article summarizes the large body of yeast studies in this field. PMID:22345607

Screening the budding yeast genome reveals unique factors affecting K2 toxin susceptibility.

PubMed

Servienė, Elena; Lukša, Juliana; Orentaitė, Irma; Lafontaine, Denis L J; Urbonavičius, Jaunius

2012-01-01

Understanding how biotoxins kill cells is of prime importance in biomedicine and the food industry. The budding yeast (S. cerevisiae) killers serve as a convenient model to study the activity of biotoxins consistently supplying with significant insights into the basic mechanisms of virus-host cell interactions and toxin entry into eukaryotic target cells. K1 and K2 toxins are active at the cell wall, leading to the disruption of the plasma membrane and subsequent cell death by ion leakage. K28 toxin is active in the cell nucleus, blocking DNA synthesis and cell cycle progression, thereby triggering apoptosis. Genome-wide screens in the budding yeast S. cerevisiae identified several hundred effectors of K1 and K28 toxins. Surprisingly, no such screen had been performed for K2 toxin, the most frequent killer toxin among industrial budding yeasts. We conducted several concurrent genome-wide screens in S. cerevisiae and identified 332 novel K2 toxin effectors. The effectors involved in K2 resistance and hypersensitivity largely map in distinct cellular pathways, including cell wall and plasma membrane structure/biogenesis and mitochondrial function for K2 resistance, and cell wall stress signaling and ion/pH homeostasis for K2 hypersensitivity. 70% of K2 effectors are different from those involved in K1 or K28 susceptibility. Our work demonstrates that despite the fact that K1 and K2 toxins share some aspects of their killing strategies, they largely rely on different sets of effectors. Since the vast majority of the host factors identified here is exclusively active towards K2, we conclude that cells have acquired a specific K2 toxin effectors set. Our work thus indicates that K1 and K2 have elaborated different biological pathways and provides a first step towards the detailed characterization of K2 mode of action.

Cleavage of precursors by the mitochondrial processing peptidase requires a compatible mature protein or an intermediate octapeptide

PubMed Central

1991-01-01

Many precursors of mitochondrial proteins are processed in two successive steps by independent matrix peptidases (MPP and MIP), whereas others are cleaved in a single step by MPP alone. To explain this dichotomy, we have constructed deletions of all or part of the octapeptide characteristic of a twice cleaved precursor (human ornithine transcarbamylase [pOTC]), have exchanged leader peptide sequences between once-cleaved (human methylmalonyl-CoA mutase [pMUT]; yeast F1ATPase beta-subunit [pF1 beta]) and twice-cleaved (pOTC; rat malate dehydrogenase (pMDH); Neurospora ubiquinol-cytochrome c reductase iron-sulfur subunit [pFe/S]) precursors, and have incubated these proteins with purified MPP and MIP. When the octapeptide of pOTC was deleted, or when the entire leader peptide of a once-cleaved precursor (pMUT or pF1 beta) was joined to the mature amino terminus of a twice-cleaved precursor (pOTC or pFe/S), no cleavage was produced by either protease. Cleavage of these constructs by MPP was restored by re- inserting as few as two amino-terminal residues of the octapeptide or of the mature amino terminus of a once-cleaved precursor. We conclude that the mature amino terminus of a twice-cleaved precursor is structurally incompatible with cleavage by MPP; such proteins have evolved octapeptides cleaved by MIP to overcome this incompatibility. PMID:1672532

[Induction of hypoxia-inducible factor-1alpha in two kinds of rats asphyxiation death models].

PubMed

Zhang, Bei-lei; Yang, Zhi-hui; Ran, Peng; Liang, Wei-bo; Zhou, Bin; Zhang, Geng-qian; Lu, Mei-li; Zhang, Lin

2007-02-15

To investigate the expression of hypoxia-inducible factor 1-alpha (HIF1-alpha) in the heart, lung, liver and kidney in rats died of two typical models of asphyxia. Two asphyxia models were made and tissue samples of the dead rats were collected from different groups at various postmortem duration. The expression and the changes of HIF1-alpha in various tissues were examined by immunohistochemistry and image analysis techniques. Results Significant expression of HIF1-alpha was observed in the myocardial fibers, kidney cells, liver cells and lung cells in both asphyxia models, but not in the control group. The expression of HIF1-alpha in various tissues in the rat died of nitrogen gas breathing was found in the nuclei at 0 hour and the expression level decreased gradually thereafter. The HIF1-alpha expression level and duration in various tissues of the rat died of hanging were higher and longer than that of the former group, with a peak of the expression level observed 6 hours after death, and then started to decline in all tissues except the heart where the expression still showed an increase 24 hours after death. The control groups showed a steady expression in the cytoplasm but not in the nuclei. HIF1-alpha appears to be a valuable biomarker in the diagnosis of asphyxia within 24 hours after death.

Smoking-related interstitial fibrosis combined with pulmonary emphysema: computed tomography-pathologic correlative study using lobectomy specimens.

PubMed

Otani, Hideji; Tanaka, Tomonori; Murata, Kiyoshi; Fukuoka, Junya; Nitta, Norihisa; Nagatani, Yukihiro; Sonoda, Akinaga; Takahashi, Masashi

2016-01-01

To evaluate the incidence and pathologic correlation of thin-section computed tomography (TSCT) findings in smoking-related interstitial fibrosis (SRIF) with pulmonary emphysema. Our study included 172 consecutive patients who underwent TSCT and subsequent lobectomy. TSCT findings including clustered cysts with visible walls (CCVW) and ground-glass attenuation with/without reticulation (GGAR) were evaluated and compared in nonsmokers and smokers and among lung locations. TSCT findings, especially CCVW, were also compared with histological findings using lobectomy specimens. The incidence of CCVW and GGAR was significantly higher in smokers than in nonsmokers (34.1% and 40.7%, respectively, vs 2.0% and 12.2%). CCVW and GGAR were frequently found in the lower and peripheral zones. Histologically, CCVW corresponded more often with SRIF with emphysema than usual interstitial pneumonia (UIP, 63.3% vs 30%). CCVW of irregular size and shape were seen in 19 of 20 SRIF with emphysema and in seven of nine UIP-manifested areas with similar round cysts. A less-involved subpleural parenchyma was observed more frequently in SRIF with emphysema. SRIF with emphysema is a more frequent pathological finding than UIP in patients with CCVW on TSCT. The irregular size and shape of CCVW and a less-involved subpleural parenchyma may be a clue suggesting the presence of SRIF with emphysema.

HPLC Analysis of [Alpha]- and [Beta]-Acids in Hops

ERIC Educational Resources Information Center

Danenhower, Travis M.; Force, Leyna J.; Petersen, Kenneth J.; Betts, Thomas A.; Baker, Gary A.

2008-01-01

Hops have been used for centuries to impart aroma and bitterness to beer. The cones of the female hop plant contain both essential oils, which include many of the fragrant components of hops, and a collection of compounds known as [alpha]- and [beta]-acids that are the precursors to bittering agents. In order for brewers to predict the ultimate…

Tumor necrosis factor-alpha stimulation of calcitonin gene-related peptide expression and secretion from rat trigeminal ganglion neurons.

PubMed

Bowen, Elizabeth J; Schmidt, Thomas W; Firm, Christina S; Russo, Andrew F; Durham, Paul L

2006-01-01

Expression of the neuropeptide calcitonin gene-related peptide (CGRP) in trigeminal ganglion is implicated in neurovascular headaches and temporomandibular joint disorders. Elevation of cytokines contributes to the pathology of these diseases. However, a connection between cytokines and CGRP gene expression in trigeminal ganglion nerves has not been established. We have focused on the effects of the cytokine tumor necrosis factor-alpha (TNF-alpha). TNFR1 receptors were found on the majority of CGRP-containing rat trigeminal ganglion neurons. Treatment of cultures with TNF-alpha stimulated CGRP secretion. In addition, the intracellular signaling intermediate from the TNFR1 receptor, ceramide, caused a similar increase in CGRP release. TNF-alpha caused a coordinate increase in CGRP promoter activity. TNF-alpha treatment activated the transcription factor NF-kappaB, as well as the Jun N-terminal kinase (JNK) and p38 mitogen-activated protein (MAP) kinase pathways. The importance of TNF-alpha induction of MAP kinase pathways was demonstrated by inhibiting MAP kinases with pharmacological reagents and gene transfer with an adenoviral vector encoding MAP kinase phosphatase-1 (MKP-1). We propose that selective and regulated inhibition of MAP kinases in trigeminal neurons may be therapeutically beneficial for inflammatory disorders involving elevated CGRP levels.

Novel Cysteine-Centered Sulfur Metabolic Pathway in the Thermotolerant Methylotrophic Yeast Hansenula polymorpha

PubMed Central

Oh, Doo-Byoung; Kwon, Ohsuk; Lee, Sang Yup; Sibirny, Andriy A.; Kang, Hyun Ah

2014-01-01

In yeast and filamentous fungi, sulfide can be condensed either with O-acetylhomoserine to generate homocysteine, the precursor of methionine, or with O-acetylserine to directly generate cysteine. The resulting homocysteine and cysteine can be interconverted through transsulfuration pathway. Here, we systematically analyzed the sulfur metabolic pathway of the thermotolerant methylotrophic yeast Hansenula polymorpha, which has attracted much attention as an industrial yeast strain for various biotechnological applications. Quite interestingly, the detailed sulfur metabolic pathway of H. polymorpha, which was reconstructed based on combined analyses of the genome sequences and validation by systematic gene deletion experiments, revealed the absence of de novo synthesis of homocysteine from inorganic sulfur in this yeast. Thus, the direct biosynthesis of cysteine from sulfide is the only pathway of synthesizing sulfur amino acids from inorganic sulfur in H. polymorpha, despite the presence of both directions of transsulfuration pathway Moreover, only cysteine, but no other sulfur amino acid, was able to repress the expression of a subset of sulfur genes, suggesting its central and exclusive role in the control of H. polymorpha sulfur metabolism. 35S-Cys was more efficiently incorporated into intracellular sulfur compounds such as glutathione than 35S-Met in H. polymorpha, further supporting the cysteine-centered sulfur pathway. This is the first report on the novel features of H. polymorpha sulfur metabolic pathway, which are noticeably distinct from those of other yeast and filamentous fungal species. PMID:24959887

Tumor Necrosis Factor alpha (TNF{alpha}) regulates CD40 expression through SMAR1 phosphorylation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Singh, Kamini; Sinha, Surajit; Malonia, Sunil Kumar

2010-01-08

CD40 plays an important role in mediating inflammatory response and is mainly induced by JAK/STAT phosphorylation cascade. TNF{alpha} is the key cytokine that activates CD40 during inflammation and tumorigenesis. We have earlier shown that SMAR1 can repress the transcription of Cyclin D1 promoter by forming a HDAC1 dependent repressor complex. In this study, we show that SMAR1 regulates the transcription of NF-{kappa}B target gene CD40. SMAR1 recruits HDAC1 and forms a repressor complex on CD40 promoter and keeps its basal transcription in check. Further, we show that TNF{alpha} stimulation induces SMAR1 phosphorylation at Ser-347 and promotes its cytoplasmic translocation, thusmore » releasing its negative effect. Concomitantly, TNF{alpha} induced phosphorylation of STAT1 at Tyr-701 by JAK1 facilitates its nuclear translocation and activation of CD40 through p300 recruitment and core Histone-3 acetylation. Thus, TNF{alpha} mediated regulation of CD40 expression occurs by dual phosphorylation of SMAR1 and STAT1.« less

Tumor necrosis factor alpha (TNF-alpha)-induced cell adhesion to human endothelial cells is under dominant control of one TNF receptor type, TNF-R55

PubMed Central

1993-01-01

Tumor necrosis factor alpha (TNF-alpha) is a pleiotropic cytokine triggering cell responses through two distinct membrane receptors. Stimulation of leukocyte adhesion to the endothelium is one of the many TNF-alpha activities and is explained by the upregulation of adhesion molecules on the endothelial cell surface. Human umbilical vein endothelial cells (HUVEC) were isolated, cultured, and demonstrated to express both TNF receptor types, TNF-R55 and TNF-R75. Cell adhesion to HUVEC was studied using the HL60, U937, and MOLT-4 cell lines. HUVEC were activated by either TNF-alpha, binding to both TNF-R55 and TNF- R75, and by receptor type-specific agonists, binding exclusively to TNF- R55 or to TNF-R75. The TNF-alpha-induced cell adhesion to HUVEC was found to be controlled almost exclusively by TNF-R55. This finding correlated with the exclusive activity of TNF-R55 in the TNF-alpha- dependent regulation of the expression of the intercellular adhesion molecule type 1 (ICAM-1), E-selectin, and vascular cell adhesion molecule type 1 (VCAM-1). The CD44 adhesion molecule in HUVEC was also found to be upregulated through TNF-R55. However, both TNF-R55 and TNF- R75 upregulate alpha 2 integrin expression in HUVEC. The predominant role of TNF-R55 in TNF-alpha-induced adhesion in HUVEC may correlate with its specific control of NF-kappa B activation, since kappa B elements are known to be present in ICAM-1, E-selectin, and VCAM-1 gene regulatory sequences. PMID:8386742

Processing of an anglerfish somatostatin precursor to a hydroxylysine-containing somatostatin 28.

PubMed Central

Spiess, J; Noe, B D

1985-01-01

A novel 28-residue somatostatin (SS) has been isolated from anglerfish pancreatic islets and characterized by complete Edman degradation, peptide mapping, and amino acid analysis. The primary structure of this anglerfish SS-28 (aSS-28) containing hydroxylysine (Hyl) was established to be H-Ser-Val-Asp-Ser-Thr-Asn-Asn-Leu-Pro-Pro-Arg-Glu-Arg-Lys-Ala-Gly-Cys- Lys-Asn-Phe-Tyr-Trp-Hyl-Gly-Phe-Thr-Ser-Cys-OH. This sequence (with the exception of hydroxylysine-23, which is replaced by lysine) is identical to the sequence of the COOH-terminal 28 residues of prepro-SS II predicted on the basis of cDNA analysis [Hobart, P., Crawford, R., Shen, L., Pictet, R. & Rutter, W. J. (1980) Nature (London) 288, 137-141]. This is the first instance in which hydroxylysine (to date characteristically observed in collagen or collagen-like structures) has been found in a potential regulatory peptide. Chromatographic characterization of peptides, radiolabeled in islet culture, revealed that aSS-28 contained 10-12% of the radioactivity incorporated into the 8000- to 1000-dalton SS-like polypeptides, whereas 88-90% of this radioactivity was detected in anglerfish SS-14. It appears probable that aSS-28 represents the predominant primary cleavage product derived from prepro-SS II by cleavage at the COOH-terminal side of a single arginine. Based on knowledge of the collagen biosynthesis, it is speculated that hydroxylation may take place as an early post-translational event. Images PMID:2857489

Nardilysin is involved in autoimmune arthritis via the regulation of tumour necrosis factor alpha secretion

PubMed Central

Fujii, Takayuki; Nishi, Eiichiro; Ito, Hiromu; Yoshitomi, Hiroyuki; Furu, Moritoshi; Okabe, Namiko; Ohno, Mikiko; Nishi, Kiyoto; Morita, Yusuke; Morita, Yugo; Azukizawa, Masayuki; Okahata, Akinori; Tomizawa, Takuya; Kimura, Takeshi; Matsuda, Shuichi

2017-01-01

Objective Tumour necrosis factor alpha (TNF-α) plays an important role in rheumatoid arthritis (RA). TNF-α is synthesised as a membrane-anchored precursor and is fully activated by a disintegrin and metalloproteinase 17 (ADAM17)-mediated ectodomain shedding. Nardilysin (NRDC) facilitates ectodomain shedding via activation of ADAM17. This study was undertaken to elucidate the role of NRDC in RA. Methods NRDC-deficient (Nrdc–/–) mice and macrophage-specific NRDC-deficient (NrdcdelM) mice were examined in murine RA models, collagen antibody-induced arthritis (CAIA) and K/BxN serum transfer arthritis (K/BxN STA). We evaluated the effect of gene deletion or silencing of Nrdc on ectodomain shedding of TNF-α in macrophages or monocytes. NRDC concentration in synovial fluid from patients with RA and osteoarthritis (OA) were measured. We also examined whether local gene silencing of Nrdc ameliorated CAIA. Results CAIA and K/BxN STA were significantly attenuated in Nrdc–/– mice and NrdcdelM mice. Gene deletion or silencing of Nrdc in macrophages or THP-1 cells resulted in the reduction of TNF-α shedding. The level of NRDC is higher in synovial fluid from RA patients compared with that from OA patients. Intra-articular injection of anti-Nrdcsmall interfering RNA ameliorated CAIA. Conclusion These data indicate that NRDC plays crucial roles in the pathogenesis of autoimmune arthritis and could be a new therapeutic target for RA treatment. PMID:28955486

Genotoxicity of diphenyl diselenide in bacteria and yeast.

PubMed

Rosa, Renato Moreira; Sulzbacher, Krisley; Picada, Jaqueline Nascimento; Roesler, Rafael; Saffi, Jenifer; Brendel, Martin; Henriques, João Antonio Pêgas

2004-10-10

Diphenyl diselenide (DPDS) is an electrophilic reagent used in the synthesis of a variety of pharmacologically active organic selenium compounds. This may increase the risk of human exposure to the chemical at the workplace. We have determined its mutagenic potential in the Salmonella/microsome assay and used the yeast Saccharomyces cerevisiae to assay for putative genotoxicity, recombinogenicity and to determine whether DNA damage produced by DPDS is repairable. Only in exponentially growing cultures was DPDS able to induce frameshift mutations in S. typhimurium and haploid yeast and to increase crossing over and gene conversion frequencies in diploid strains of S. cerevisiae. Thus, DPDS presents a behavior similar to that of an intercalating agent. Mutants defective in excision-resynthesis repair (rad3, rad1), in error-prone repair (rad6) and in recombinational repair (rad52) showed higher than WT-sensitivity to DPDS. It appears that this compound is capable of inducing single and/or double strand breaks in DNA. An epistatic interaction was shown between rad3-e5 and rad52-1 mutant alleles, indicating that excision-resynthesis and strand-break repair may possess common steps in the repair of DNA damage induced by DPDS. DPDS was able to enhance the mutagenesis induced by oxidative mutagens in bacteria. N-acetylcysteine, a glutathione biosynthesis precursor, prevented mutagenesis induced by DPDS in yeast. We have shown that DPDS is a weak mutagen which probably generates DNA strand breaks through both its intercalating action and pro-oxidant effect.

Physiological and environmental control of yeast prions

PubMed Central

Chernova, Tatiana A.; Wilkinson, Keith D.; Chernoff, Yury O.

2014-01-01

Prions are self-perpetuating protein isoforms that cause fatal and incurable neurodegenerative disease in mammals. Recent evidence indicates that a majority of human proteins involved in amyloid and neural inclusion disorders possess at least some prion properties. In lower eukaryotes, such as yeast, prions act as epigenetic elements, which increase phenotypic diversity by altering a range of cellular processes. While some yeast prions are clearly pathogenic, it is also postulated that prion formation could be beneficial in variable environmental conditions. Yeast and mammalian prions have similar molecular properties. Crucial cellular factors and conditions influencing prion formation and propagation were uncovered in the yeast models. Stress-related chaperones, protein quality control deposits, degradation pathways and cytoskeletal networks control prion formation and propagation in yeast. Environmental stresses trigger prion formation and loss, supposedly acting via influencing intracellular concentrations of the prion-inducing proteins, and/or by localizing prionogenic proteins to the prion induction sites via heterologous ancillary helpers. Physiological and environmental modulation of yeast prions points to new opportunities for pharmacological intervention and/or prophylactic measures targeting general cellular systems rather than the properties of individual amyloids and prions. PMID:24236638

The development of novel inhibitors of tumor necrosis factor-alpha production based on substituted [5,5]-bicyclic pyrozolones

DOE Office of Scientific and Technical Information (OSTI.GOV)

Laufersweiler, Matthew; Brugel, Todd; Clark, Michael

Novel substituted [5,5]-bicyclic pyrzazolones are presented as inhibitors of tumor necrosis factor-{alpha} (TNF-{alpha}) production. Many of these compounds show low nanomolar activity against lipopolysaccaride (LPS)-induced TNF-{alpha} production in THP-1 cells. This class of molecules was co-crystallized with mutated p38, and several analogs showed good oral bioavailability in the rat. Oral activity of these compounds in the rat iodoacetate model for osteoarthritis is discussed.

Persistent tumor necrosis factor signaling in normal human fibroblasts prevents the complete resynthesis of I kappa B-alpha.

PubMed

Poppers, D M; Schwenger, P; Vilcek, J

2000-09-22

Transcription factor NF-kappa B is normally sequestered in the cytoplasm, complexed with I kappa B inhibitory proteins. Tumor necrosis factor (TNF) and interleukin-1 induce I kappa B-alpha phosphorylation, leading to I kappa B-alpha degradation and translocation of NF-kappa B to the nucleus where it activates genes important in inflammatory and immune responses. TNF and interleukin-1 actions are typically terminated by desensitization, and I kappa B-alpha reappearance normally occurs within 30-60 min. We found that in normal human FS-4 fibroblasts maintained in the presence of TNF, I kappa B-alpha protein failed to return to base-line levels for up to 15 h. Removal of TNF at any time during the 15-h period resulted in complete I kappa B-alpha resynthesis, suggesting that I kappa B-alpha reappearance was prevented by continued TNF signaling. Long term exposure of FS-4 fibroblasts to TNF led to a persistent presence of I kappa B-alpha mRNA, sustained I kappa B kinase activation, continuous proteasome-mediated degradation of I kappa B-alpha, and sustained nuclear localization of NF-kappa B. Continuous exposure of FS-4 cells to TNF did not lead to a sustained activation of p38 or ERK mitogen-activated protein kinases, suggesting that not all TNF-induced signaling pathways are persistently activated. These findings challenge the notion that all cytokine-mediated signals are rapidly terminated by desensitization and illustrate the need to elucidate the process of deactivation of TNF-induced signaling.

Purification and characterisation of a new hypothalamic satiety peptide, cocaine and amphetamine regulated transcript (CART), produced in yeast.

PubMed

Thim, L; Nielsen, P F; Judge, M E; Andersen, A S; Diers, I; Egel-Mitani, M; Hastrup, S

1998-05-29

Cocaine and amphetamine regulated transcript (CART) is a newly discovered hypothalamic peptide with a potent appetite suppressing activity following intracerebroventricular administration. When the mature rat CART sequence encoding CART(1-102) was inserted in the yeast expression plasmid three CART peptides could be purified from the fermentation broth reflecting processing at dibasic sequences. None of these corresponded to the naturally occurring CART(55-102). In order to obtain CART(55-102) the precursor Glu-Glu-Ile-Asp-CART(55-102) has been produced and CART(55-102) was generated by digestion of the precursor with dipeptidylaminopeptidase-1. All four generated CART peptides have been characterised by N-terminal amino acid sequencing and mass spectrometry. The CART peptides contain six cysteine residues and using the yeast expressed CART(62-102) the disulphide bond configuration was found to be I-III, II-V and IV-VI. When the four CART peptides were intracerebroventricularly injected in fasted mice (0.1 to 2.0 microg) they all produced a dose dependent inhibition of food intake.

In vitro protective effects of two extracts from bergamot peels on human endothelial cells exposed to tumor necrosis factor-alpha (TNF-alpha).

PubMed

Trombetta, Domenico; Cimino, Francesco; Cristani, Mariateresa; Mandalari, Giuseppina; Saija, Antonella; Ginestra, Giovanna; Speciale, Antonio; Chirafisi, Joselita; Bisignano, Giuseppe; Waldron, Keith; Narbad, Arjan; Faulds, Craig B

2010-07-28

Bergamot ( Citrus bergamia Risso) is a less commercialized Citrus fruit, mainly used for its essential oil extracted from the peel. Bergamot peel (BP) represents about 60% of the processed fruits and is regarded as primary waste. However, it contains good amounts of useful compounds, such as pectins and flavonoids. Many of the bioactivities of Citrus flavonoids appear to impact vascular endothelial cells. Herein, we report the protective effect of two flavonoid-rich extracts from BP (endowed with radical-scavenging properties and lacking genotoxic activity) against alterations in cell modifications induced by the pleiotropic inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) on human umbilical vein endothelial cells (HUVECs), as demonstrated by monitoring intracellular levels of malondialdehyde/4-hydroxynonenal, reduced and oxidized glutathione and superoxide dismutase activity, and the activation status of nuclear factor-kappaB (NF-kappaB). Thus, BP appears to be a potential source of natural antioxidant/anti-inflammatory phytocomplexes to be employed as ingredients of nutraceutical products or functional foods.

The influence of tumour necrosis factor-alpha on the cardiovascular system of anaesthetized rats.

PubMed

Tabrizchi, R

2001-03-01

The effects of two vasoactive agents (adenosine A2A agonist, CGS 21680, and adrenoceptor agonist, noradrenaline) were examined on cardiac output (CO), heart rate (HR), blood pressure (BP), mean circulatory filling pressure (Pmcf), resistance to venous return, arterial resistance, dP/dt, plasma levels of NO2-/NO3-, and inducible nitric oxide synthase (iNOS) activity in lungs ex vivo, following treatment with tumour necrosis factor-alpha (TNF-alpha; 30 microg/kg) in anaesthetized rats. Treatment with TNF-alpha produced significant reduction in CO (41+/-2%), dP/dt (26+/-3%), BP (26+/-2%) and Pmcf (27+/-4%; n=6; mean+/-SEM), but increased arterial resistance. There were no significant changes in the plasma levels of NO2-/NO3-levels over time following treatment with TNF-alpha, but there was a significant increase (approximately twofold) in the activity of the iNOS in the lungs of animals treated with TNF-alpha. Administration of CGS 21680 (1.0 microg/kg per min) significantly increased CO (44+/-6%), HR (12+/-2%), Pmcf (24+/-4%) and dP/dt (24+/-5%) in TNF-alpha-treated rats. CGS 21680 also significantly reduced arterial resistance (33+/-2%) without altering resistance to venous return in TNF-alpha-treated rats. While noradrenaline (1.0 microg/kg per min) infusion did not significantly increase CO, it did significantly increase HR (12+/-1%), BP (55+/-9%), Pmcf (47+/-5%), dP/dt (65+/-7%), resistance to venous return (64+/-20%), and arterial resistance (41+/-16%) in TNF-alpha-treated animals. The reduction in BP due to administration of TNF-alpha is the result of significant reduction in CO. Consequently, the decline in CO can be attributed to a combination of a negative inotropic effect as well as a reduction in Pmcf. It is evident that infusion with CGS 21680 could reverse the negative impact of TNF-alpha on CO by increasing dP/dt, Pmcf and HR as well as a reduction in arterial resistance. The fact that noradrenaline did not significantly increase CO in TNF-alpha

The Interrelationships of Mathematical Precursors in Kindergarten

PubMed Central

Cirino, Paul T.

2011-01-01

This study evaluated the interrelations among cognitive precursors across quantitative, linguistic, and spatial attention domains that have been implicated for math achievement in young children. The dimensionality of the quantity precursors was evaluated in 286 Kindergarteners via latent variable techniques, and the contribution of precursors from each domain was established for small sums addition. Results showed a five factor structure for the quantity precursors with the major distinction between nonsymbolic and symbolic tasks. The overall model demonstrated good fit, and strong predictive power (R2 = 55%) for addition number combinations. Linguistic and spatial attention domains showed indirect relationships with outcomes, with their effects mediated by symbolic quantity measures. These results have implications for the measurement of mathematical precursors, and yield promise for predicting future math performance. PMID:21194711

Calcium alpha-ketoglutarate administration to malnourished hemodialysis patients improves plasma arginine concentrations.

PubMed

Riedel, E; Hampl, H; Steudle, V; Nündel, M

1996-01-01

Calcium alpha-ketoglutarate administration to 24 malnourished hemodialysis patients for 1 year leads to a significant increase in plasma concentrations of L-arginine from 53.6 +/- 18.3 (compared to a healthy control group: 87.5 +/- 27.3) to 71.1 +/- 15.9 mumol/l (p < 0.05). Furthermore, concentrations in plasma of proline and histidine, precursors of glutamate biosynthesis, are increased; inorganic phosphate and urea are significantly decreased in hemodialysis patients after 1 year of calcium alpha-ketoglutarate administration.

Extracellular Polysaccharides Produced by Yeasts and Yeast-Like Fungi

NASA Astrophysics Data System (ADS)

van Bogaert, Inge N. A.; de Maeseneire, Sofie L.; Vandamme, Erick J.

Several yeasts and yeast-like fungi are known to produce extracellular polysaccharides. Most of these contain D-mannose, either alone or in combination with other sugars or phosphate. A large chemical and structural variability is found between yeast species and even among different strains. The types of polymers that are synthesized can be chemically characterized as mannans, glucans, phosphoman-nans, galactomannans, glucomannans and glucuronoxylomannans. Despite these differences, almost all of the yeast exopolysaccharides display some sort of biological activity. Some of them have already applications in chemistry, pharmacy, cosmetics or as probiotic. Furthermore, some yeast exopolysaccharides, such as pullulan, exhibit specific physico-chemical and rheological properties, making them useful in a wide range of technical applications. A survey is given here of the production, the characteristics and the application potential of currently well studied yeast extracellular polysaccharides.

Novel characteristics of the biological properties of the yeast Saccharomyces cerevisiae eukaryotic initiation factor 2A.

PubMed

Komar, Anton A; Gross, Stephane R; Barth-Baus, Diane; Strachan, Ryan; Hensold, Jack O; Goss Kinzy, Terri; Merrick, William C

2005-04-22

Eukaryotic initiation factor 2A (eIF2A) has been shown to direct binding of the initiator methionyl-tRNA (Met-tRNA(i)) to 40 S ribosomal subunits in a codon-dependent manner, in contrast to eIF2, which requires GTP but not the AUG codon to bind initiator tRNA to 40 S subunits. We show here that yeast eIF2A genetically interacts with initiation factor eIF4E, suggesting that both proteins function in the same pathway. The double eIF2A/eIF4E-ts mutant strain displays a severe slow growth phenotype, which correlated with the accumulation of 85% of the double mutant cells arrested at the G(2)/M border. These cells also exhibited a disorganized actin cytoskeleton and elevated actin levels, suggesting that eIF2A might be involved in controlling the expression of genes involved in morphogenic processes. Further insights into eIF2A function were gained from the studies of eIF2A distribution in ribosomal fractions obtained from either an eIF5BDelta (fun12Delta) strain or a eIF3b-ts (prt1-1) strain. It was found that the binding of eIF2A to 40 and 80 S ribosomes was not impaired in either strain. We also found that eIF2A functions as a suppressor of Ure2p internal ribosome entry site-mediated translation in yeast cells. The regulation of expression from the URE2 internal ribosome entry site appears to be through the levels of eIF2A protein, which has been found to be inherently unstable with a half-life of approximately 17 min. It was hypothesized that this instability allows for translational control through the level of eIF2A protein in yeast cells.

Free hemoglobin enhances tumor necrosis factor-alpha production in isolated human monocytes.

PubMed

Carrillo, Eddy H; Gordon, Laura E; Richardson, J David; Polk, Hiram C

2002-03-01

A systemic inflammatory response (SIR) is seen in approximately 75% of patients with complex blunt liver injuries treated nonoperatively. Many feel this response is caused by blood, bile, and necrotic tissue accumulation in the peritoneal cavity. Our current treatment for these patients is a delayed laparoscopic washout of the peritoneal cavity, resulting in a dramatic resolution of the SIR. Spectrophotometric analysis of the intraperitoneal fluid has confirmed the presence of high concentrations of free hemoglobin (Hb). We hypothesize that free Hb enhances the local peritoneal response by increasing tumor necrosis factor-alpha (TNF-alpha) production by monocytes, contributing to the local inflammatory response and SIR. Monocytes from five healthy volunteers were isolated and cultured in RPMI-1640 for 24 hours. Treatment groups included saline controls, lipopolysaccharide ([LPS], 10 ng/mL, from Escherichia coli), human Hb (25 microg/mL), and Hb + LPS. Supernatants were analyzed by enzyme-linked immunosorbent assay. Student's t test with Mann-Whitney posttest was used for statistical analysis with p < or = 0.05 considered significant. Free Hb significantly increased TNF-alpha production 915 +/- 223 pg/mL versus saline (p = 0.02). LPS and Hb + LPS further increased TNF-alpha production (2294 pg/mL and 2501 pg/mL, respectively, p < 0.001) compared with saline controls. These data confirm that free Hb is a proinflammatory mediator resulting in the production of significant amounts of TNF-alpha. These in vitro findings support our clinical data in which timely removal of intraperitoneal free hemoglobin helps prevent its deleterious local and systemic inflammatory effects in patients with complex liver injuries managed nonoperatively.

Yeast Infection (Vaginal)

MedlinePlus

Yeast infection (vaginal) Overview A vaginal yeast infection is a fungal infection that causes irritation, discharge and intense itchiness ... symptoms Causes The fungus candida causes a vaginal yeast infection. Your vagina naturally contains a balanced mix of yeast, including ...

Contractile-ring assembly in fission yeast cytokinesis: Recent advances and new perspectives.

PubMed

Lee, I-Ju; Coffman, Valerie C; Wu, Jian-Qiu

2012-10-01

The fission yeast Schizosaccharomyces pombe is an excellent model organism to study cytokinesis. Here, we review recent advances on contractile-ring assembly in fission yeast. First, we summarize the assembly of cytokinesis nodes, the precursors of a normal contractile ring. IQGAP Rng2 and myosin essential light chain Cdc4 are recruited by the anillin-like protein Mid1, followed by the addition of other cytokinesis node proteins. Mid1 localization on the plasma membrane is stabilized by interphase node proteins. Second, we discuss proteins and processes that contribute to the search, capture, pull, and release mechanism of contractile-ring assembly. Actin filaments nucleated by formin Cdc12, the motor activity of myosin-II, the stiffness of the actin network, and severing of actin filaments by cofilin all play essential roles in contractile-ring assembly. Finally, we discuss the Mid1-independent pathway for ring assembly, and the possible mechanisms underlying the ring maturation and constriction. Collectively, we provide an overview of the current understanding of contractile-ring assembly and uncover future directions in studying cytokinesis in fission yeast. Copyright © 2012 Wiley Periodicals, Inc.

The Yeast Copper Response Is Regulated by DNA Damage

PubMed Central

Dong, Kangzhen; Addinall, Stephen G.; Lydall, David

2013-01-01

Copper is an essential but potentially toxic redox-active metal, so the levels and distribution of this metal are carefully regulated to ensure that it binds to the correct proteins. Previous studies of copper-dependent transcription in the yeast Saccharomyces cerevisiae have focused on the response of genes to changes in the exogenous levels of copper. We now report that yeast copper genes are regulated in response to the DNA-damaging agents methyl methanesulfonate (MMS) and hydroxyurea by a mechanism(s) that requires the copper-responsive transcription factors Mac1 and AceI, copper superoxide dismutase (Sod1) activity, and the Rad53 checkpoint kinase. Furthermore, in copper-starved yeast, the response of the Rad53 pathway to MMS is compromised due to a loss of Sod1 activity, consistent with the model that yeast imports copper to ensure Sod1 activity and Rad53 signaling. Crucially, the Mac1 transcription factor undergoes changes in its redox state in response to changing levels of copper or MMS. This study has therefore identified a novel regulatory relationship between cellular redox, copper homeostasis, and the DNA damage response in yeast. PMID:23959798

Role of tumour necrosis factor alpha converting enzyme (TACE/ADAM17) and associated proteins in coronary artery disease and cardiac events.

PubMed

Chemaly, Melody; McGilligan, Victoria; Gibson, Mark; Clauss, Matthias; Watterson, Steven; Alexander, H Denis; Bjourson, Anthony John; Peace, Aaron

2017-12-01

Tumour necrosis factor alpha converting enzyme (TACE/ADAM17) is a member of the A disintegrin and metalloproteinase (ADAM) family of ectodomain shedding proteinases. It regulates many inflammatory processes by cleaving several transmembrane proteins, including tumour necrosis factor alpha (TNFα) and its receptors tumour necrosis factor alpha receptor 1 and tumour necrosis factor alpha receptor 2. There is evidence that TACE is involved in several inflammatory diseases, such as ischaemia, heart failure, arthritis, atherosclerosis, diabetes and cancer as well as neurological and immune diseases. This review summarizes the latest discoveries regarding the mechanism of action and regulation of TACE. It also focuses on the role of TACE in atherosclerosis and coronary artery disease (CAD), highlighting clinical studies that have investigated its expression and protein activity. The multitude of substrates cleaved by TACE make this enzyme an attractive target for therapy and a candidate for biomarker research and development in CAD. Crown Copyright © 2017. Published by Elsevier Masson SAS. All rights reserved.

Tumor necrosis factor-alpha and interleukin-4 gene polymorphisms in Chinese patients with gout.

PubMed

Chen, M-L; Tsai, F-J; Tsai, C-H; Huang, C-M

2007-01-01

The purpose of this study was to examine whether polymorphisms of interleukin-4 (IL-4) (promoter-590 and intron 3) and tumor necrosis factor-alpha (TNF-alpha) promoter-308 genes are markers of susceptibility to or clinical manifestations of gout in Taiwanese patients. The study included 196 Taiwanese patients with gout and 103 unrelated healthy control subjects living in central Taiwan. Polymorphisms of the IL-4 (promoter-590 and intron 3) and TNF-alpha (promoter-308) genes were typed from genomic DNA. Allelic frequencies and carriage rates were then compared between gout patients and control subjects. The correlation between allelic frequencies, carriage rates and clinical manifestations of gout were evaluated. No significant differences were observed in the allelic frequencies and carriage rates of the IL-4 (promoter-590 and intron 3) and TNF-alpha gene polymorphisms between patients with gout and healthy control subjects. Furthermore, the IL-4 (promoter-590 and intron 3) and TNF-alpha genotypes were not found to be associated with the clinical and laboratory profiles in gout patients. However, there was a significant difference in the TNF-alphapolymorphism genotype between patients with and without hypertriglyceridemia (P=0.001, xi2=11.47, OR=10.3, 95%CI=3.57-29.7). The results of our study suggest that polymorphisms of the IL-4 (promoter-590 and intron 3) and TNF-alpha promoter-308 genes are not related to gout in Chinese patients in Taiwan.

Immunological and biochemical characterization of processing products from the neurotensin/neuromedin N precursor in the rat medullary thyroid carcinoma 6-23 cell line.

PubMed Central

Bidard, J N; de Nadai, F; Rovere, C; Moinier, D; Laur, J; Martinez, J; Cuber, J C; Kitabgi, P

1993-01-01

Neurotensin (NT) and neuromedin N (NN) are two related biologically active peptides that are encoded in the same precursor molecule. In the rat, the precursor consists of a 169-residue polypeptide starting with an N-terminal signal peptide and containing in its C-terminal region one copy each of NT and NN. NN precedes NT and is separated from it by a Lys-Arg sequence. Two other Lys-Arg sequences flank the N-terminus of NN and the C-terminus of NT. A fourth Lys-Arg sequence occurs near the middle of the precursor and is followed by an NN-like sequence. Finally, an Arg-Arg pair is present within the NT moiety. The four Lys-Arg doublets represent putative processing sites in the precursor molecule. The present study was designed to investigate the post-translational processing of the NT/NN precursor in the rat medullary thyroid carcinoma (rMTC) 6-23 cell line, which synthesizes large amounts of NT upon dexamethasone treatment. Five region-specific antisera recognizing the free N- or C-termini of sequences adjacent to the basic doublets were produced, characterized and used for immunoblotting and radioimmunoassay studies in combination with gel filtration, reverse-phase h.p.l.c. and trypsin digestion of rMTC 6-23 cell extracts. Because two of the antigenic sequences, i.e. NN and the NN-like sequence, start with a lysine residue that is essential for recognition by their respective antisera, a micromethod by which trypsin specifically cleaves at arginine residues was developed. The results show that dexamethasone-treated rMTC 6-23 cells produced comparable amounts of NT, NN and a peptide corresponding to a large N-terminal precursor fragment lacking the NN and NT moieties. This large fragment was purified. N-Terminal sequencing revealed that it started at residue Ser23 of the prepro-NT/NN sequence, and thus established the Cys22-Ser23 bond as the cleavage site of the signal peptide. Two other large N-terminal fragments bearing respectively the NN and NT sequences at

[Malassezia yeasts and their significance in dermatology].

PubMed

Hort, W; Nilles, M; Mayser, P

2006-07-01

Yeasts of the genus Malassezia belong to the normal microflora of the human skin. In addition they are known to cause a variety of skin diseases; the most frequent of which is pityriasis versicolor. Malassezia yeasts are also thought to be associated with seborrheic dermatitis, dandruff and Malassezia folliculitis. Recently the significance of Malassezia yeasts as a trigger factor for atopic dermatitis of the head and neck region has been pointed out. The role of the Malassezia yeasts in these different diseases has been controversial in the past and remains an issue because of difficulties in isolation, culture and differentiation of the organism. Thanks to molecular techniques, 10 species can actually be differentiated. The article presents the different Malassezia-associated diseases, their clinical picture, diagnosis and appropriate therapy. In addition the speciation of Malassezia is reviewed.

Alternative function for the mitochondrial SAM complex in biogenesis of alpha-helical TOM proteins.

PubMed

Stojanovski, Diana; Guiard, Bernard; Kozjak-Pavlovic, Vera; Pfanner, Nikolaus; Meisinger, Chris

2007-12-03

The mitochondrial outer membrane contains two preprotein translocases: the general translocase of outer membrane (TOM) and the beta-barrel-specific sorting and assembly machinery (SAM). TOM functions as the central entry gate for nuclear-encoded proteins. The channel-forming Tom40 is a beta-barrel protein, whereas all Tom receptors and small Tom proteins are membrane anchored by a transmembrane alpha-helical segment in their N- or C-terminal portion. Synthesis of Tom precursors takes place in the cytosol, and their import occurs via preexisting TOM complexes. The precursor of Tom40 is then transferred to SAM for membrane insertion and assembly. Unexpectedly, we find that the biogenesis of alpha-helical Tom proteins with a membrane anchor in the C-terminal portion is SAM dependent. Each SAM protein is necessary for efficient membrane integration of the receptor Tom22, whereas assembly of the small Tom proteins depends on Sam37. Thus, the substrate specificity of SAM is not restricted to beta-barrel proteins but also includes the majority of alpha-helical Tom proteins.

Effect of long-term exposure to mobile phone radiation on alpha-Int1 gene sequence of Candida albicans.

PubMed

Shahin-Jafari, Ariyo; Bayat, Mansour; Shahhosseiny, Mohammad Hassan; Tajik, Parviz; Roudbar-Mohammadi, Shahla

2016-05-01

Over the last decade, communication industries have witnessed a tremendous expansion, while, the biological effects of electromagnetic waves have not been fully elucidated. Current study aimed at evaluating the mutagenic effect of long-term exposure to 900-MHz radiation on alpha-Int1 gene sequences of Candida albicans. A standard 900 MHz radiation generator was used for radiation. 10 ml volumes from a stock suspension of C. albicans were transferred into 10 polystyrene tubes. Five tubes were exposed at 4 °C to a fixed magnitude of radiation with different time periods of 10, 70, 210, 350 and 490 h. The other 5 tubes were kept far enough from radiation. The samples underwent genomic DNA extraction. PCR amplification of alpha-Int1 gene sequence was done using one set of primers. PCR products were resolved using agarose gel electrophoresis and the nucleotide sequences were determined. All samples showed a clear electrophoretic band around 441 bp and further sequencing revealed the amplified DNA segments are related to alpha-Int1 gene of the yeast. No mutations in the gene were seen in radiation exposed samples. Long-term exposure of the yeast to mobile phone radiation under the above mentioned conditions had no mutagenic effect on alpha-Int1 gene sequence.

Impaired coactivator activity of the Gly{sub 482} variant of peroxisome proliferator-activated receptor {gamma} coactivator-1{alpha} (PGC-1{alpha}) on mitochondrial transcription factor A (Tfam) promoter

DOE Office of Scientific and Technical Information (OSTI.GOV)

Choi, Yon-Sik; Hong, Jung-Man; Lim, Sunny

2006-06-09

Mitochondrial dysfunction may cause diabetes or insulin resistance. Peroxisome proliferation-activated receptor-{gamma} (PPAR-{gamma}) coactivator-1 {alpha} (PGC-1{alpha}) increases mitochondrial transcription factor A (Tfam) resulting in mitochondrial DNA content increase. An association between a single nucleotide polymorphism (SNP), G1444A(Gly482Ser), of PGC-1{alpha} coding region and insulin resistance has been reported in some ethnic groups. In this study, we investigated whether a change of glycine to serine at codon 482 of PGC-1{alpha} affected the Tfam promoter activity. The cDNA of PGC-1{alpha} variant bearing either glycine or serine at 482 codon was transfected into Chang human hepatocyte cells. The PGC-1{alpha} protein bearing glycine had impaired coactivatormore » activity on Tfam promoter-mediated luciferase. We analyzed the PGC-1{alpha} genotype G1444A and mitochondrial DNA (mtDNA) copy number from 229 Korean leukocyte genomic DNAs. Subjects with Gly/Gly had a 20% lower amount of peripheral blood mtDNA than did subjects with Gly/Ser and Ser/Ser (p < 0.05). No correlation was observed between diabetic parameters and PGC-1{alpha} genotypes in Koreans. These results suggest that PGC-1{alpha} variants with Gly/Gly at 482nd amino acid may impair the Tfam transcription, a regulatory function of mitochondrial biogenesis, resulting in dysfunctional mtDNA replication.« less

The Budding Yeast Nucleus

PubMed Central

Taddei, Angela; Schober, Heiko; Gasser, Susan M.

2010-01-01

The budding yeast nucleus, like those of other eukaryotic species, is highly organized with respect to both chromosomal sequences and enzymatic activities. At the nuclear periphery interactions of nuclear pores with chromatin, mRNA, and transport factors promote efficient gene expression, whereas centromeres, telomeres, and silent chromatin are clustered and anchored away from pores. Internal nuclear organization appears to be function-dependent, reflecting localized sites for tRNA transcription, rDNA transcription, ribosome assembly, and DNA repair. Recent advances have identified new proteins involved in the positioning of chromatin and have allowed testing of the functional role of higher-order chromatin organization. The unequal distribution of silent information regulatory factors and histone modifying enzymes, which arises in part from the juxtaposition of telomeric repeats, has been shown to influence chromatin-mediated transcriptional repression. Other localization events suppress unwanted recombination. These findings highlight the contribution budding yeast genetics and cytology have made to dissecting the functional role of nuclear structure. PMID:20554704

Distinct Domestication Trajectories in Top-Fermenting Beer Yeasts and Wine Yeasts.

PubMed

Gonçalves, Margarida; Pontes, Ana; Almeida, Pedro; Barbosa, Raquel; Serra, Marta; Libkind, Diego; Hutzler, Mathias; Gonçalves, Paula; Sampaio, José Paulo

2016-10-24

Beer is one of the oldest alcoholic beverages and is produced by the fermentation of sugars derived from starches present in cereal grains. Contrary to lager beers, made by bottom-fermenting strains of Saccharomyces pastorianus, a hybrid yeast, ale beers are closer to the ancient beer type and are fermented by S. cerevisiae, a top-fermenting yeast. Here, we use population genomics to investigate (1) the closest relatives of top-fermenting beer yeasts; (2) whether top-fermenting yeasts represent an independent domestication event separate from those already described; (3) whether single or multiple beer yeast domestication events can be inferred; and (4) whether top-fermenting yeasts represent non-recombinant or recombinant lineages. Our results revealed that top-fermenting beer yeasts are polyphyletic, with a main clade composed of at least three subgroups, dominantly represented by the German, British, and wheat beer strains. Other beer strains were phylogenetically close to sake, wine, or bread yeasts. We detected genetic signatures of beer yeast domestication by investigating genes previously linked to brewing and using genome-wide scans. We propose that the emergence of the main clade of beer yeasts is related with a domestication event distinct from the previously known cases of wine and sake yeast domestication. The nucleotide diversity of the main beer clade more than doubled that of wine yeasts, which might be a consequence of fundamental differences in the modes of beer and wine yeast domestication. The higher diversity of beer strains could be due to the more intense and different selection regimes associated to brewing. Copyright © 2016 Elsevier Ltd. All rights reserved.

[Treatment of oil-manufacturing wastewater by yeast-SBR system].

PubMed

Lü, Wen-zhou; Liu, Ying; Huang, Yi-zhen

2008-04-01

Eight yeast strains were applied to a sequencing batch reactor (SBR) to treat high-strength oil-containing wastewater. The removal performance, yeast cultivation method and key factors affecting the stability of system were discussed. The results show yeast sludge with MLSS of 19 g/L and SVI of 35 mL/g can be obtained in 6 d in an open system without any molds and bacteria inhibitor addition; In 30 d continuous wastewater treatment, COD and oil removal rate achieve 86.8%-96.9% and above 99.5% respectively under the influent conditions of the COD of 9000-23000 mg/L and oil of 4500-16000 mg/L; Short period of pH impact brings reversible effects on the system and the sludge retention time can affect the SVI of the yeast; Absence of nitrogen induces morphology conversion of some yeast cells from single cell to filamentous one and impairs the settling capability of the yeast.

Hypoxia-inducible factor 1-alpha release after intracoronary versus intramyocardial stem cell therapy in myocardial infarction.

PubMed

Gyöngyösi, Mariann; Hemetsberger, Rayyan; Posa, Aniko; Charwat, Silvia; Pavo, Noemi; Petnehazy, Ors; Petrasi, Zsolt; Pavo, Imre J; Hemetsberger, Hani; Benedek, Imre; Benedek, Teodora; Benedek, Istvan; Kovacs, Istvan; Kaun, Christoph; Maurer, Gerald

2010-04-01

We have investigated the effect of stem cell delivery on the release of hypoxia-inducible factor 1 alpha (HIF-1alpha) in peripheral circulation and myocardium in experimental myocardial ischemia. Closed-chest, reperfused myocardial infarction (MI) was created in domestic pigs. Porcine mesenchymal stem cells (MSCs) were cultured and delivered (9.8 +/- 1.2 x 10(6)) either percutaneously NOGA-guided transendocardially (Group IM) or intracoronary (Group IC) 22 +/- 4 days post-MI. Pigs without MSC delivery served as sham control (Group S). Plasma HIF-1alpha was measured at baseline, immediately post- and at follow-up (FUP; 2 h or 24 h) post-MSC delivery by ELISA kit. Myocardial HIF-1alpha expression of infarcted, normal myocardium, or border zone was determined by Western blot. Plasma level of HIF-1alpha increased immediately post-MI (from 278 +/- 127 to 631 +/- 375 pg/ml, p < 0.05). Cardiac delivery of MSCs elevated the plasma levels of HIF-1alpha significantly (p < 0.05) in groups IC and IM immediately post-MSC delivery, and returned to baseline level at FUP, without difference between the groups IC and IM. The myocardial tissue HIF-1alpha expression in the infarcted area was higher in Group IM than in Group IC or S (1,963 +/- 586 vs. 1,307 +/- 392 vs. 271 +/- 110 activity per square millimeter, respectively, p < 0.05), while the border zone contained similarly lower level of HIF-1alpha, but still significantly higher as compared with Group S. Trend towards increase in myocardial expression of HIF-1alpha was measured in Group IM at 24 h, in contrast to Group IC. In conclusion, both stem cell delivery modes increase the systemic and myocardial level of HIF-1alpha. Intramyocardial delivery of MSC seems to trigger the release of angiogenic HIF-1alpha more effectively than does intracoronary delivery.

Metabolism of 4-Chloronitrobenzene by the Yeast Rhodosporidium sp

PubMed Central

Corbett, Michael D.; Corbett, Bernadette R.

1981-01-01

The yeast Rhodosporidium sp. metabolized 4-chloronitrobenzene by a reductive pathway to give 4-chloroacetanilide and 4-chloro-2-hydroxyacetanilide as the major final metabolites. The intermediate production of 4-chloronitrosobenzene, 4-chlorophenylhydroxylamine, and 4-chloroaniline was demonstrated by high-pressure liquid chromatography. Additional studies with selected metabolites established that the metabolite 4-chloro-2-hydroxyacetanilide was produced by an initial Bamberger rearrangement of the hydroxylamine metabolite, followed by acetylation. Direct C hydroxylation of the aromatic ring was not observed in this species. No hydroxamic acid production was detected, even though significant concentrations of the nitroso and hydroxylamine precursors to this functional group were observed. PMID:16345757

Tumour necrosis factor-alpha blockers: potential limitations in the management of advanced endometriosis? A case report.

PubMed

Shakiba, Khashayar; Falcone, Tommaso

2006-09-01

Several studies have shown that tumour necrosis factor (TNF)-alpha levels are increased in the peritoneal fluid of women with endometriosis, with correlation between TNF-alpha concentrations and the degree of disease. It is also likely that elevation of peritoneal fluids' TNF-alpha levels may play a role in the pathogenesis of infertility associated with endometriosis. Use of drugs such as etanercept, a TNF-alpha receptor immunoglobulin fusion protein which inhibits TNF-alpha activity, showed in an animal study to reduce the severity of the disease, and the size of endometriotic foci. TNF-alpha blockers were recommended as a possible new line of therapy for endometriosis. Our case involved a 35-year-old Para 0, with rheumatic arthritis and stage 4 endometriosis. After 6 years of constant use of etanercept, she showed no improvement of endometriosis as demonstrated at laparoscopy. However, she underwent a successful IVF after the first attempt. TNF-alpha-blocker medications might not be beneficial for patients with advanced endometriosis. However, we cannot exclude the possible effect of these medications on early-stage endometriosis, and further study is required. Some of the immunologic abnormalities in the pelvis of patients with endometriosis could be the consequence of the disease and not the cause, and possibly suppression of immune cells and their products may not have a major effect on endometriotic lesions at an advanced stage. This also could explain why suppression of TNF-alpha showed no effect on infertility. However, use of TNF-alpha-blockers before IVF might increase the success rate in advanced endometriosis.

Negative feedback regulation of human platelets via autocrine activation of the platelet-derived growth factor alpha-receptor.

PubMed

Vassbotn, F S; Havnen, O K; Heldin, C H; Holmsen, H

1994-05-13

Human platelets contain platelet-derived growth factor (PDGF) in their alpha-granules which is released during platelet exocytosis. We show by immunoprecipitation and 125I-PDGF binding experiments that human platelets have functionally active PDGF alpha-receptors, but not beta-receptors. The PDGF alpha-receptor (PDGFR-alpha) was identified as a 170-kDa glycosylated protein-tyrosine kinase as found in other cell types. Stimulation of platelets with 0.1 unit/ml thrombin resulted in a significant increase (2-5-fold) of the tyrosine phosphorylation of the PDGFR-alpha, as determined by immunoprecipitation with phosphotyrosine antiserum as well as with PDGFR-alpha antiserum. The observed thrombin-induced autophosphorylation of the PDGFR-alpha was inhibited by the addition of a neutralizing monoclonal PDGF antibody. Thus, our results suggest that the platelet PDGFR-alpha is stimulated in an autocrine manner by PDGF secreted during platelet activation. Preincubation of platelets with PDGF inhibited thrombin-induced platelet aggregation and secretion of ATP + ADP and beta-hexosaminidase. Thrombin-induced platelet aggregation was also reversed when PDGF was added 30 s after thrombin stimulation. Inhibition of the autocrine PDGF pathway during platelet activation by the PDGF antibody led to a potentiation of thrombin-induced beta-hexosaminidase secretion. Thus, the PDGFR-alpha takes part in a negative feedback regulation during platelet activation. Our demonstration of PDGF alpha-receptors on human platelets and its inhibitory function during platelet activation identifies a new possible role of PDGF in the regulation of thrombosis.

Smoking-related interstitial fibrosis combined with pulmonary emphysema: computed tomography-pathologic correlative study using lobectomy specimens

PubMed Central

Otani, Hideji; Tanaka, Tomonori; Murata, Kiyoshi; Fukuoka, Junya; Nitta, Norihisa; Nagatani, Yukihiro; Sonoda, Akinaga; Takahashi, Masashi

2016-01-01

Purpose To evaluate the incidence and pathologic correlation of thin-section computed tomography (TSCT) findings in smoking-related interstitial fibrosis (SRIF) with pulmonary emphysema. Patients and methods Our study included 172 consecutive patients who underwent TSCT and subsequent lobectomy. TSCT findings including clustered cysts with visible walls (CCVW) and ground-glass attenuation with/without reticulation (GGAR) were evaluated and compared in nonsmokers and smokers and among lung locations. TSCT findings, especially CCVW, were also compared with histological findings using lobectomy specimens. Results The incidence of CCVW and GGAR was significantly higher in smokers than in nonsmokers (34.1% and 40.7%, respectively, vs 2.0% and 12.2%). CCVW and GGAR were frequently found in the lower and peripheral zones. Histologically, CCVW corresponded more often with SRIF with emphysema than usual interstitial pneumonia (UIP, 63.3% vs 30%). CCVW of irregular size and shape were seen in 19 of 20 SRIF with emphysema and in seven of nine UIP-manifested areas with similar round cysts. A less-involved subpleural parenchyma was observed more frequently in SRIF with emphysema. Conclusion SRIF with emphysema is a more frequent pathological finding than UIP in patients with CCVW on TSCT. The irregular size and shape of CCVW and a less-involved subpleural parenchyma may be a clue suggesting the presence of SRIF with emphysema. PMID:27445472

Mga2 Transcription Factor Regulates an Oxygen-responsive Lipid Homeostasis Pathway in Fission Yeast*

PubMed Central

Burr, Risa; Stewart, Emerson V.; Shao, Wei; Zhao, Shan; Hannibal-Bach, Hans Kristian; Ejsing, Christer S.; Espenshade, Peter J.

2016-01-01

Eukaryotic lipid synthesis is oxygen-dependent with cholesterol synthesis requiring 11 oxygen molecules and fatty acid desaturation requiring 1 oxygen molecule per double bond. Accordingly, organisms evaluate oxygen availability to control lipid homeostasis. The sterol regulatory element-binding protein (SREBP) transcription factors regulate lipid homeostasis. In mammals, SREBP-2 controls cholesterol biosynthesis, whereas SREBP-1 controls triacylglycerol and glycerophospholipid biosynthesis. In the fission yeast Schizosaccharomyces pombe, the SREBP-2 homolog Sre1 regulates sterol homeostasis in response to changing sterol and oxygen levels. However, notably missing is an SREBP-1 analog that regulates triacylglycerol and glycerophospholipid homeostasis in response to low oxygen. Consistent with this, studies have shown that the Sre1 transcription factor regulates only a fraction of all genes up-regulated under low oxygen. To identify new regulators of low oxygen adaptation, we screened the S. pombe nonessential haploid deletion collection and identified 27 gene deletions sensitive to both low oxygen and cobalt chloride, a hypoxia mimetic. One of these genes, mga2, is a putative transcriptional activator. In the absence of mga2, fission yeast exhibited growth defects under both normoxia and low oxygen conditions. Mga2 transcriptional targets were enriched for lipid metabolism genes, and mga2Δ cells showed disrupted triacylglycerol and glycerophospholipid homeostasis, most notably with an increase in fatty acid saturation. Indeed, addition of exogenous oleic acid to mga2Δ cells rescued the observed growth defects. Together, these results establish Mga2 as a transcriptional regulator of triacylglycerol and glycerophospholipid homeostasis in S. pombe, analogous to mammalian SREBP-1. PMID:27053105

Low susceptibility of NC/Nga mice to tumor necrosis factor-alpha-mediated lethality and hepatocellular damage with D-galactosamine sensitization.

PubMed

Koide, Naoki; Morikawa, Akiko; Naiki, Yoshikazu; Tumurkhuu, Gantsetseg; Yoshida, Tomoaki; Ikeda, Hiroshi; Yokochi, Takashi

2009-02-01

The susceptibility of NC/Nga mice to tumor necrosis factor (TNF)-alpha was examined by using sensitization with d-galactosamine (d-GalN). Administration of TNF-alpha and d-GalN killed none of the NC/Nga mice, whereas it killed all of the BALB/c mice. Treatment with TNF-alpha and d-GalN caused few hepatic lesions in NC/Nga mice but massive hepatocellular apoptosis in BALB/c mice. Unlike BALB/c mice, there was no elevation in caspase 3 and 8 activities in the livers of NC/Nga mice receiving TNF-alpha and d-GalN. On the other hand, administration of anti-Fas antibody definitely killed both NC/Nga and BALB/c mice via activation of caspases 3 and 8. Treatment with TNF-alpha and d-GalN led to translocation of nuclear factor (NF)-kappaB in NC/Nga and BALB/c mice. However, NF-kappaB translocation was sustained in NC/Nga mice, although it disappeared in BALB/c mice 7 h after the treatment. NF-kappaB inhibitors activated caspases 3 and 8, and enhanced TNF-alpha-mediated lethality in NC/Nga. Taken together, the low susceptibility of NC/Nga mice to TNF-alpha-mediated lethality was suggested to be responsible for the sustained NF-kappaB activation.

Role of hypoxia-inducible factor-{alpha} in hepatitis-B-virus X protein-mediated MDR1 activation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Han, Hyo-Kyung; Han, Chang Yeob; Cheon, Eun-Pa

2007-06-01

The transition from chemotherapy-responsive cancer cells to chemotherapy-resistant cancer cells is mainly accompanied by the increased expression of multi-drug resistance 1 (MDR1). We found that hepatitis-B-virus X protein (HBx) increases the transcriptional activity and protein level of MDR1 in a hepatoma cell line, H4IIE. In addition, HBx overexpression made H4IIE cells more resistant to verapamil-uptake. HBx stabilized hypoxia-inducible factor-1{alpha} (HIF-1{alpha}) and induced the nuclear translocation of C/EBP{beta}. Reporter gene analyses showed that HBx increased the reporter activity in the cells transfected with the reporter containing MDR1 gene promoter. Moreover, the luciferase reporter gene activity was significantly inhibited by HIF-1{alpha} siRNAmore » but not by overexpression of C/EBP dominant negative mutant. These results imply that HBx increases the MDR1 transporter activity through the transcriptional activation of the MDR1 gene with HIF-1{alpha} activation, and suggest HIF-1{alpha} for the therapeutic target of HBV-mediated chemoresistance.« less

Anhydrobiosis in yeast: cell wall mannoproteins are important for yeast Saccharomyces cerevisiae resistance to dehydration.

PubMed

Borovikova, Diana; Teparić, Renata; Mrša, Vladimir; Rapoport, Alexander

2016-08-01

The state of anhydrobiosis is linked with the reversible delay of metabolism as a result of strong dehydration of cells, and is widely distributed in nature. A number of factors responsible for the maintenance of organisms' viability in these conditions have been revealed. This study was directed to understanding how changes in cell wall structure may influence the resistance of yeasts to dehydration-rehydration. Mutants lacking various cell wall mannoproteins were tested to address this issue. It was revealed that mutants lacking proteins belonging to two structurally and functionally unrelated groups (proteins non-covalently attached to the cell wall, and Pir proteins) possessed significantly lower cell resistance to dehydration-rehydration than the mother wild-type strain. At the same time, the absence of the GPI-anchored cell wall protein Ccw12 unexpectedly resulted in an increase of cell resistance to this treatment; this phenomenon is explained by the compensatory synthesis of chitin. The results clearly indicate that the cell wall structure/composition relates to parameters strongly influencing yeast viability during the processes of dehydration-rehydration, and that damage to cell wall proteins during yeast desiccation can be an important factor leading to cell death. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Three-Dimensional Conformal Radiotherapy in Prostate Cancer Patients: Rise in Interleukin 6 (IL-6) but not IL-2, IL-4, IL-5, Tumor Necrosis Factor-{alpha}, MIP-1-{alpha}, and LIF Levels

DOE Office of Scientific and Technical Information (OSTI.GOV)

Oliveira Lopes, Carlos; Callera, Fernando, E-mail: fcallera@gmail.com

Purpose: To investigate the effect of radiotherapy (RT) on serum levels of interleukin-2 (IL-2), IL-4, IL-5, IL-6, tumor necrosis factor alpha (TNF-{alpha}), macrophage inflammatory protein-1-alpha (MIP-1-{alpha}) and leukemia inhibitory factor (LIF) in patients with prostate cancer. Methods and Materials: Forty eight patients with prostate cancer received three-dimensional conformal blocking radiation therapy with a linear accelerator. IL-2, IL-4, IL-5, IL-6, TNF-{alpha}, MIP-1-{alpha}, and LIF levels were measured by the related immunoassay kit 1 day before the beginning of RT and during RT at days 15 and 30. Results: The mean IL-2 values were elevated before and during the RT in contrastmore » with those of IL-4, IL-5, IL-6, TNF-{alpha}, MIP-1-{alpha}, and LIF, which were within the normal range under the same conditions. Regarding markers IL-2, IL-4, IL-5, TNF-{alpha}, MIP-1-{alpha}, and LIF, comparisons among the three groups (before treatment and 15 and 30 days during RT) did not show significant differences. Although values were within the normal range, there was a significant rise in IL-6 levels at day 15 of RT (p = 0.0049) and a decline at day 30 to levels that were similar to those observed before RT. Conclusions: IL-6 appeared to peak after 15 days of RT before returning to pre-RT levels. In contrast, IL-2, IL-4, IL-5, TNF-{alpha}, MIP-1-{alpha}, and LIF levels were not sensitive to irradiation. The increased levels of IL-6 following RT without the concurrent elevation of other cytokines involved in the acute phase reaction did not suggest a classical inflammatory response to radiation exposure. Further studies should be designed to elucidate the role of IL-6 levels in patients with prostate cancer treated with RT.« less

Human monocytes and gingival fibroblasts release tumor necrosis factor-alpha, interleukin-1 alpha and interleukin-6 in response to particulate and soluble fractions of Prevotella melaninogenica and Fusobacterium nucleatum.

PubMed

Rossano, F; Rizzo, A; Sanges, M R; Cipollaro de L'Ero, G; Tufano, M A

1993-01-01

In this study we provide evidence that structural and soluble components of periodontopathogenic bacteria, such as Prevotella melaninogenica and Fusobacterium nucleatum, induce the release of cytokines in vitro known to cause in vivo necrotic inflammatory phenomena and bone resorption (tumor necrosis factor-alpha, interleukin-1 alpha and interleukin-6). Human monocytes and gingival fibroblasts were cultivated in vitro in the presence of both particulate and soluble bacterial fractions. A dose-dependent production of tumor necrosis factor-alpha by monocytes and gingival fibroblasts was observed in the presence of fractions of P. melaninogenica and F. nucleatum. Interleukin-1 alpha was produced in approximately the same quantities in the presence of soluble fractions of either P. melaninogenica or F. nucleatum, but in greater quantities in response to particulate fractions of P. melaninogenica. Monocytes released larger amounts of interleukin-1 alpha (about 3000 pg/ml) than gingival fibroblasts (about 1500 pg/ml). Interleukin-6 was released in greater quantities by monocytes in the presence of the pellet fraction of P. melaninogenica (about 5.5 ng/ml), but gingival fibroblasts released larger amounts of interleukin-6, especially in the presence of particulate and soluble components of F. nucleatum (about 12 ng/ml). The ability to induce the release of these cytokines notably increases the pathogenic potential of the bacteria involved in the damage of periodontal tissue.

Effect of temperature on replicative aging of the budding yeast Saccharomyces cerevisiae.

PubMed

Molon, Mateusz; Zadrag-Tecza, Renata

2016-04-01

The use of the budding yeast Saccharomyces cerevisiae in gerontological studies was based on the assumption that the reproduction limit of a single cell (replicative aging) is a consequence of accumulation of a hypothetical universal "senescence factor" within the mother cell. However, some evidence suggests that molecules or structures proposed as the "aging factor", such as rDNA circles, oxidatively damaged proteins (with carbonyl groups) or mitochondria, have little effect on replicative lifespan of yeast cells. Our results also suggest that protein aggregates associated with Hsp104, treated as a marker of yeast aging, do not seem to affect the numeric value of replicative lifespan of yeast. What these results indicate, however, is the need for finding a different way of expressing age and longevity of yeast cells instead of the commonly used number of daughters produced over units of time, as in the case of other organisms. In this paper, we show that the temperature has a stronger influence on the time of life (the total lifespan) than on the reproductive potential of yeast cells.

Inhibitory effects of Turkish folk remedies on inflammatory cytokines: interleukin-1alpha, interleukin-1beta and tumor necrosis factor alpha.

PubMed

Yeşilada, E; Ustün, O; Sezik, E; Takaishi, Y; Ono, Y; Honda, G

1997-09-01

In this study, in vitro inhibitory effects of 55 extracts or fractions obtained from 10 plant species on interleukin-1 (IL-1alpha, IL-1beta) and tumor necrosis factor (TNF-alpha) biosynthesis were studied. The following plant materials from Turkish folk medicine for the treatment of various diseases which are thought to be inflammatory in nature e.g. rheumatism, fever, infections, edemas or related inflammatory diseases were selected as the subject of this study: Cistus laurifolius leaves, Clematis flammna flowering herbs, Crataegus orientalis roots, Daphne oleoides ssp. oleoides whole plant, Ecbalium elaterium roots, Rosa canina roots, Rubus discolor roots, Rubus hirtus roots, Sambucus ebulus flowers and leaves, Sambucus nigra flowers and leaves. All plants showed inhibitory activity against at least one of these models in various percentages depending upon the concentration, thus supporting the folkloric utilization. Daphne oleoides was found to be the most active plant against the test models.

[Cardiovascular exercise on obese women: effects on adiponectine, leptine, and tumour necrosis factor-alpha].

PubMed

Landeros-Olvera, Erick; López-Alvarenga, Juan Carlos; Nava-González, Edna J; Gallegos-Cabriales, Esther; Lavalle-González, Fernando; Bastarrachea, Raúl A; Salazar González, Bertha Cecilia

2014-01-01

The relationship of hormones adiponectin, leptin and tumor necrosis factor-alpha in adipose tissue on the atherogenic process is one of the most promising models in preventive medicine. The numerous tests performed to identify the effect of exercise on these hormones have not been clear on the type of exercise routine and physical effort calculated to contribute to changing plasma concentrations in obese women. Analyze controlledcardiovascular exercise effect on serum level of adiponectin, leptin, and tumournecrosis factor-alpha in obese young women. A simple blind clinical essay. The intervention covered a 10-week controlled, cardiovascular exercise program by 34 women (cases n=17, controls n=17) with a body mass index>27kg/m(2). Molecular analysis was performed by immune-fluorescence. Following the intervention, cases and controls means were as follows: adiponectin 19.0 vs. 12.2μ/ml (P=.008); leptin 20.0 vs. 28.0μ/L (P=.02); and tumour necrosis factor-alpha 4.7 vs. 5.1pg/ml (P=.05). The established exercise (5 sessions a week of exercise of 40min each for 10 weeks with a heart rate reserve of 40 to 80%) improved plasma concentrations of these hormones in the expected direction. This finding highlights an unpublished amount of exercise, controlled by the reserve cardiac frequency that might contribute the cardiovascular and metabolic protection to obese women. Copyright © 2013 Instituto Nacional de Cardiología Ignacio Chávez. Published by Masson Doyma México S.A. All rights reserved.

Cloning of the prepro C-RFa gene and brain localization of the active peptide in Salmo salar.

PubMed

Montefusco-Siegmund, R A; Romero, A; Kausel, G; Muller, M; Fujimoto, M; Figueroa, J

2006-08-01

In all vertebrates, the synthesis and release of prolactin (Prl) from pituitary lactotroph cells is tightly controlled by hypothalamic factors. We have cloned and characterized a hypothalamic cDNA from Atlantic salmon (Salmo salar) encoding C-RFa, a peptide structurally related to mammalian Prl-releasing peptide (PrRP). The deduced preprohormone precursor is composed of 155 amino acid residues presenting a 87.1% similarity to chum salmon C-RFa and a 100% similarity to all fish C-RFa in the bioactive precursor motifs. C-RFa-immunoreactive perikarya and fibres were located in the brain of S. salar, especially in the hypothalamus, olfactory tract, optic tectum and cerebellum. In contrast, immunolabelled fibres were not observed in the pituitary stalk or in the hypophysis. However, interestingly, we detected immunolabelled cells in the rostral pars distalis of the pituitary in the basolateral region in which Prl is synthesized. These results were confirmed by obtaining a strong signal by using reverse transcription/polymerase chain reaction (RT-PCR) on mRNA from both hypothalamus and pituitary. These data show, for the first time, by immunohistochemistry and RT-PCR, that C-RFa is produced in pituitary cells. Finally, based on these results, a possible function for C-RFa as a locally produced PrRP in this teleost is discussed.

Development of a mouse-feline chimeric antibody against feline tumor necrosis factor-alpha.

PubMed

Doki, Tomoyoshi; Takano, Tomomi; Hohdatsu, Tsutomu

2016-10-01

Feline infectious peritonitis (FIP) is a fatal inflammatory disease caused by FIP virus infection. Feline tumor necrosis factor (fTNF)-alpha is closely involved in the aggravation of FIP pathology. We previously described the preparation of neutralizing mouse anti-fTNF-alpha monoclonal antibody (mAb 2-4) and clarified its role in the clinical condition of cats with FIP using in vitro systems. However, administration of mouse mAb 2-4 to cat may lead to a production of feline anti-mouse antibodies. In the present study, we prepared a mouse-feline chimeric mAb (chimeric mAb 2-4) by fusing the variable region of mouse mAb 2-4 to the constant region of feline antibody. The chimeric mAb 2-4 was confirmed to have fTNF-alpha neutralization activity. Purified mouse mAb 2-4 and chimeric mAb 2-4 were repeatedly administered to cats, and the changes in the ability to induce feline anti-mouse antibody response were investigated. In the serum of cats treated with mouse mAb 2-4, feline anti-mouse antibody production was induced, and the fTNF-alpha neutralization effect of mouse mAb 2-4 was reduced. In contrast, in cats treated with chimeric mAb 2-4, the feline anti-mouse antibody response was decreased compared to that of mouse mAb 2-4-treated cats.

The Innate Lymphoid Cell Precursor.

PubMed

Ishizuka, Isabel E; Constantinides, Michael G; Gudjonson, Herman; Bendelac, Albert

2016-05-20

The discovery of tissue-resident innate lymphoid cell populations effecting different forms of type 1, 2, and 3 immunity; tissue repair; and immune regulation has transformed our understanding of mucosal immunity and allergy. The emerging complexity of these populations along with compounding issues of redundancy and plasticity raise intriguing questions about their precise lineage relationship. Here we review advances in mapping the emergence of these lineages from early lymphoid precursors. We discuss the identification of a common innate lymphoid cell precursor characterized by transient expression of the transcription factor PLZF, and the lineage relationships of innate lymphoid cells with conventional natural killer cells and lymphoid tissue inducer cells. We also review the rapidly growing understanding of the network of transcription factors that direct the development of these lineages.

Molecular characterization of variant alpha-subunit of electron transfer flavoprotein in three patients with glutaric acidemia type II--and identification of glycine substitution for valine-157 in the sequence of the precursor, producing an unstable mature protein in a patient.

PubMed Central

Indo, Y; Glassberg, R; Yokota, I; Tanaka, K

1991-01-01

In our previous study of eight glutaric acidemia type II (GAII) fibroblast lines by using [35S]methionine labeling and immunoprecipitation, three of them had a defect in the synthesis of the alpha-subunit of electron transfer flavoprotein (alpha-ETF) (Ikeda et al. 1986). In one of them (YH1313) the labeling of the mature alpha-ETF was barely detectable, while that of the precursor (p) was stronger. In another (YH605) no synthesis of immunoreactive p alpha-ETF was detectable. In the third cell line (YH1391) the rate of variant p alpha-ETF synthesis was comparable to normal, but its electrophoretic mobility was slightly faster than normal. In the present study, the northern blot analysis revealed that all three mutant cell lines contained p alpha-ETF mRNA and that their size and amount were comparable to normal. In immunoblot analysis, both alpha- and beta-ETF bands were barely detectable in YH1313 and YH605 but were detectable in YH1391 in amounts comparable to normal. Sequencing of YH1313 p alpha-ETF cDNA via PCR identified a transversion of T-470 to G. We then devised a simple PCR method for the 119-bp section (T-443/G-561) for detecting this mutation. In the upstream primer, A-466 was artificially replaced with C, to introduce a BstNI site into the amplified copies in the presence of G-470 from the variant sequence. The genomic DNA analysis using this method demonstrated that YH1313 was homozygous for T----G-470 transversion. It was not detected either in two other alpha-ETF-deficient GAII or in seven control cell lines. The alpha-ETF cDNA sequence in YH605 was identical to normal. Images Figure 1 Figure 2 Figure 3 Figure 5 PMID:1882842

ADP-ribosylation factor arf6p may function as a molecular switch of new end take off in fission yeast

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fujita, Atsushi

2008-02-01

Small GTPases act as molecular switches in a wide variety of cellular processes. In fission yeast Schizosaccharomyces pombe, the directions of cell growth change from a monopolar manner to a bipolar manner, which is known as 'New End Take Off' (NETO). Here I report the identification of a gene, arf6{sup +}, encoding an ADP-ribosylation factor small GTPase, that may be essential for NETO. arf6{delta} cells completely fail to undergo NETO. arf6p localizes at both cell ends and presumptive septa in a cell-cycle dependent manner. And its polarized localization is not dependent on microtubules, actin cytoskeletons and some NETO factors (bud6p,more » for3p, tea1p, tea3p, and tea4p). Notably, overexpression of a fast GDP/GTP-cycling mutant of arf6p can advance the timing of NETO. These findings suggest that arf6p functions as a molecular switch for the activation of NETO in fission yeast.« less

Nutritional regulation of hepatic heme biosynthesis and porphyria through PGC-1alpha.

PubMed

Handschin, Christoph; Lin, Jiandie; Rhee, James; Peyer, Anne-Kathrin; Chin, Sherry; Wu, Pei-Hsuan; Meyer, Urs A; Spiegelman, Bruce M

2005-08-26

Inducible hepatic porphyrias are inherited genetic disorders of enzymes of heme biosynthesis. The main clinical manifestations are acute attacks of neuropsychiatric symptoms frequently precipitated by drugs, hormones, or fasting, associated with increased urinary excretion of delta-aminolevulinic acid (ALA). Acute attacks are treated by heme infusion and glucose administration, but the mechanisms underlying the precipitating effects of fasting and the beneficial effects of glucose are unknown. We show that the rate-limiting enzyme in hepatic heme biosynthesis, 5-aminolevulinate synthase (ALAS-1), is regulated by the peroxisome proliferator-activated receptor gamma coactivator 1alpha (PGC-1alpha). Elevation of PGC-1alpha in mice via adenoviral vectors increases the levels of heme precursors in vivo as observed in acute attacks. The induction of ALAS-1 by fasting is lost in liver-specific PGC-1alpha knockout animals, as is the ability of porphyrogenic drugs to dysregulate heme biosynthesis. These data show that PGC-1alpha links nutritional status to heme biosynthesis and acute hepatic porphyria.

Screening the Budding Yeast Genome Reveals Unique Factors Affecting K2 Toxin Susceptibility

PubMed Central

Servienė, Elena; Lukša, Juliana; Orentaitė, Irma

2012-01-01

Background Understanding how biotoxins kill cells is of prime importance in biomedicine and the food industry. The budding yeast (S. cerevisiae) killers serve as a convenient model to study the activity of biotoxins consistently supplying with significant insights into the basic mechanisms of virus-host cell interactions and toxin entry into eukaryotic target cells. K1 and K2 toxins are active at the cell wall, leading to the disruption of the plasma membrane and subsequent cell death by ion leakage. K28 toxin is active in the cell nucleus, blocking DNA synthesis and cell cycle progression, thereby triggering apoptosis. Genome-wide screens in the budding yeast S. cerevisiae identified several hundred effectors of K1 and K28 toxins. Surprisingly, no such screen had been performed for K2 toxin, the most frequent killer toxin among industrial budding yeasts. Principal Findings We conducted several concurrent genome-wide screens in S. cerevisiae and identified 332 novel K2 toxin effectors. The effectors involved in K2 resistance and hypersensitivity largely map in distinct cellular pathways, including cell wall and plasma membrane structure/biogenesis and mitochondrial function for K2 resistance, and cell wall stress signaling and ion/pH homeostasis for K2 hypersensitivity. 70% of K2 effectors are different from those involved in K1 or K28 susceptibility. Significance Our work demonstrates that despite the fact that K1 and K2 toxins share some aspects of their killing strategies, they largely rely on different sets of effectors. Since the vast majority of the host factors identified here is exclusively active towards K2, we conclude that cells have acquired a specific K2 toxin effectors set. Our work thus indicates that K1 and K2 have elaborated different biological pathways and provides a first step towards the detailed characterization of K2 mode of action. PMID:23227207

Thalidomide suppressed interleukin-6 but not tumor necrosis factor-alpha in volunteers with experimental endotoxemia.

PubMed

Shannon, Edward; Noveck, Robert; Sandoval, Felipe; Kamath, Burde; Kearney, Michael

2007-11-01

An early rationale for using thalidomide to treat erythema nodosum leprosum had been based on some reports that it suppresses tumor necrosis factor-alpha (TNF-alpha). However, in vivo and in vitro studies have yielded variable results, having shown that thalidomide can either enhance or suppress TNF-alpha. Since the course of circulating cytokines like TNF-alpha after infusion of endotoxin into volunteers is reproducible and characteristic, we investigated the effect of thalidomide on endotoxin-induced synthesis of TNF-alpha, interleukin (IL)-6, and IL-8. The cytokine response from 18 placebo-treated subjects who had undergone the endotoxin challenge were pooled with a placebo-treated subject from the current study and were compared with 4 subjects who received thalidomide (100 mg) every 6 h for 5 doses before endotoxin challenge. Thirty minutes after the last dose of thalidomide or placebo, volunteers were infused with 4-ng/kg endotoxin. Plasma was collected and assayed for cytokines by enzyme-linked immunosorbent assay. Endotoxin evoked the synthesis of the cytokines in all volunteers. The peak response for TNF-alpha was 1.5 h, 2.5 h for IL-8, and 3.0 h for IL-6. Thalidomide did not significantly delay the release of cytokines into the circulating blood. At the peak response, thalidomide reduced the concentration of the cytokines in the plasma. Using the area under the dose response curve (AUC(0 to 24) h), thalidomide reduced the AUC for IL-6 by 56%, for IL-8 by 30%, and TNF-alpha by 32%. In this model, thalidomide did not suppress TNF-alpha or IL-8, but it did suppress IL-6 at 4-h postinfusion with lipopolysaccharide (P=0.004), at 6 h (P=0.014), at 12 h (P=0.001), and at 16 h (P=0.012).

Efficient expression and purification of recombinant therapeutic protein candidates, human midkine and pleiotrophin.

PubMed

Murasugi, Akira

2013-01-01

Midkine is a heparin-binding growth factor that promotes cell growth, survival, and migration. Externally added midkine prevents ventricular remodeling and improves long-term survival after myocardial infarction in the mouse. Preclinical testing of this protein is in progress. Externally added pleiotrophin, a member of the midkine protein family, promotes functional recovery after neural transplantation in rats. Thus, pleiotrophin is also a candidate therapeutic protein. Large amounts of these proteins were obtained by using the heterologous protein expression system of Pichia pastoris, and the recombinant P. pastoris clones were cultured in a controlled fermentor. Intracellular expression yielded about 300 mg/L recombinant human (rh)-midkine, which was extracted, renatured, and purified. From 1 L of the culture, 64 mg of rh-midkine was purified. Secretory expression induced by the midkine secretion signal resulted in about 100 mg of rhmidkine in 1 L of the culture supernatant, but over 70% of the rh-midkine had yeast-specific glycosylation. Three threonyl residues that are targets for glycosylation were substituted with alanyl residues, and nonglycosylated, active rh-midkine was obtained. In secretory expression using α-mating factor prepro-sequence, about 640 mg/L rh-midkine was obtained, but it was partially truncated. Therefore, a protease-deficient host was used, and about 360 mg/L intact rh-midkine was then obtained. The rh-midkine was recovered and purified, with 70% final yield. All purified rh-midkine, regardless of expression method, was able to promote mammalian cell proliferation. In secretory expression of rh-pleiotrophin using α- mating factor prepro-sequence, 260 mg/L rh-pleiotrophin could be secreted. The rh-pleiotrophin was recovered and efficiently purified with 72% final yield.

Ceramide does not mediate the effect of tumour necrosis factor alpha on superoxide generation in human neutrophils.

PubMed Central

Yanaga, F; Watson, S P

1994-01-01

The effect of tumour necrosis factor alpha (TNF alpha) on superoxide generation in human neutrophils was investigated using the Nitro Blue Tetrazolium reduction assay. TNF alpha stimulated superoxide generation in a time- and concentration-dependent fashion. The maximally effective concentration of TNF alpha for superoxide generation was 10 nM and maximal response was obtained after 15-20 min. The monoclonal antibody (mAb), utr-1, which was raised against the 75 kDa receptor and behaves as an antagonist, had no effect on superoxide generation, but partially inhibited the response to TNF alpha. mAb htr-9, which was raised against the 55 kDa receptor and behaves as an agonist, mimicked the effect of TNF alpha, but with a lower maximal response. As it has been reported that ceramide might act as a second messenger to mediate many of the effects of TNF alpha, the effects of exogenous sphingomyelinase and the cell-permeable ceramide analogue, C2- ceramide, on production of superoxide anions, induction of priming in response to formylmethionyl-leucyl-phenylalanine, and cell-shape change were examined. Neither sphingomyelinase nor C2-ceramide mimicked the effect of TNF alpha. Ceramide is converted into ceramide 1-phosphate by ceramide kinase and we have measured levels of this metabolite to clarify the effect of TNF alpha on sphingomyelinase activity in neutrophils. Although exogenous sphingomyelinase increased the amount of ceramide 1-phosphate in a time-dependent manner, and C2-ceramide was rapidly converted into C2-ceramide phosphate, TNF alpha had no effect on the level of ceramide 1-phosphate. These results suggest that TNF alpha stimulates superoxide generation through both the 55 kDa and 75 kDa receptors, but that ceramide does not act as an intracellular mediator for TNF alpha in human neutrophils. Images Figure 4 PMID:8141790

New Complexity-Building Reactions of Alpha-Keto Esters

NASA Astrophysics Data System (ADS)

Bartlett, Samuel L.

I. Introduction: Importance of Asymmetric Catalysis and the Reactivity Patterns of alpha-Keto Esters. II. Synthesis of Complex Tertiary Glycolates by Enantioconvergent Arylation of Stereochemically Labile alpha-Keto Esters. Enantioconvergent arylation reactions of boronic acids and racemic ?-stereogenic alpha-keto esters have been developed. The reactions are catalyzed by a chiral (diene)Rh(I) complex and provide a wide array of beta-stereogenic tertiary aryl glycolate derivatives with high levels of diastereo- and enantioselectivity. Racemization studies employing a series of sterically differentiated tertiary amines suggest that the steric nature of the amine base additive exerts a significant influence on the rate of substrate racemization. III. Palladium-Catalyzed beta-Arylation of alpha-Keto Esters . A catalyst system derived from commercially available Pd2(dba) 3 and PtBu3 has been applied to the coupling of alpha-keto ester enolates and aryl bromides. The reaction provides access to an array of beta-stereogenic alpha-keto ester derivatives. When the air stable ligand precursor PtBu 3˙HBF4 is employed, the reaction can be carried out without use of a glovebox. The derived products are of broad interest given the prevalence of the alpha-keto acid substructure in biologically important molecules. IV. Catalytic Enantioselective [3+2] Cycloaddition of alpha-Keto Ester Enolates and Nitrile Oxides. An enantioselective [3+2] cycloaddition reaction between nitrile oxides and transiently generated enolates of alpha-keto esters has been developed. The catalyst system was found to be compatible with in situ nitrile oxide generation conditions. A versatile array of nitrile oxides and alpha-keto esters could participate in the cycloaddition, providing novel 5-hydroxy-2-isoxazolines in high chemical yield with high levels of diastereo- and enantioselectivity. Notably, the optimal reaction conditions circumvented concurrent reaction via O-imidoylation and hetero-[3

Drosophila Regulate Yeast Density and Increase Yeast Community Similarity in a Natural Substrate

PubMed Central

Stamps, Judy A.; Yang, Louie H.; Morales, Vanessa M.; Boundy-Mills, Kyria L.

2012-01-01

Drosophila melanogaster adults and larvae, but especially larvae, had profound effects on the densities and community structure of yeasts that developed in banana fruits. Pieces of fruit exposed to adult female flies previously fed fly-conditioned bananas developed higher yeast densities than pieces of the same fruits that were not exposed to flies, supporting previous suggestions that adult Drosophila vector yeasts to new substrates. However, larvae alone had dramatic effects on yeast density and species composition. When yeast densities were compared in pieces of the same fruits assigned to different treatments, fruits that developed low yeast densities in the absence of flies developed significantly higher yeast densities when exposed to larvae. Across all of the fruits, larvae regulated yeast densities within narrow limits, as compared to a much wider range of yeast densities that developed in pieces of the same fruits not exposed to flies. Larvae also affected yeast species composition, dramatically reducing species diversity across fruits, reducing variation in yeast communities from one fruit to the next (beta diversity), and encouraging the consistent development of a yeast community composed of three species of yeast (Candida californica, C. zemplinina, and Pichia kluvyeri), all of which were palatable to larvae. Larvae excreted viable cells of these three yeast species in their fecal pools, and discouraged the growth of filamentous fungi, processes which may have contributed to their effects on the yeast communities in banana fruits. These and other findings suggest that D. melanogaster adults and their larval offspring together engage in ‘niche construction’, facilitating a predictable microbial environment in the fruit substrates in which the larvae live and develop. PMID:22860093

Effect of long-term exposure to mobile phone radiation on alpha-Int1 gene sequence of Candida albicans

PubMed Central

Shahin-jafari, Ariyo; Bayat, Mansour; Shahhosseiny, Mohammad Hassan; Tajik, Parviz; Roudbar-mohammadi, Shahla

2015-01-01

Over the last decade, communication industries have witnessed a tremendous expansion, while, the biological effects of electromagnetic waves have not been fully elucidated. Current study aimed at evaluating the mutagenic effect of long-term exposure to 900-MHz radiation on alpha-Int1 gene sequences of Candida albicans. A standard 900 MHz radiation generator was used for radiation. 10 ml volumes from a stock suspension of C. albicans were transferred into 10 polystyrene tubes. Five tubes were exposed at 4 °C to a fixed magnitude of radiation with different time periods of 10, 70, 210, 350 and 490 h. The other 5 tubes were kept far enough from radiation. The samples underwent genomic DNA extraction. PCR amplification of alpha-Int1 gene sequence was done using one set of primers. PCR products were resolved using agarose gel electrophoresis and the nucleotide sequences were determined. All samples showed a clear electrophoretic band around 441 bp and further sequencing revealed the amplified DNA segments are related to alpha-Int1 gene of the yeast. No mutations in the gene were seen in radiation exposed samples. Long-term exposure of the yeast to mobile phone radiation under the above mentioned conditions had no mutagenic effect on alpha-Int1 gene sequence. PMID:27081370

On the difficulties of detecting PP precursors

NASA Astrophysics Data System (ADS)

Lessing, Stephan; Thomas, Christine; Saki, Morvarid; Schmerr, Nicholas; Vanacore, Elizabeth

2015-06-01

The PP precursors are seismic waves that form from underside reflections of P waves off discontinuities in the upper mantle transition zone (MTZ). These seismic phases are used to map discontinuity topography, sharpness, and impedance contrasts; the resulting structural variations are then often interpreted as evidence for temperature and/or mineralogy variations within the mantle. The PP precursors as well as other seismic phases have been used to establish the global presence of seismic discontinuities at 410 and 660 km depth. Intriguingly, in more than 80 per cent of PP precursor observations the seismic wave amplitudes are significantly weaker than the amplitudes predicted by seismic reference models. Even more perplexing is the observation that 1-5 per cent of all earthquakes (which are 20-25 per cent of earthquakes with clear PP waveforms) do not show any evidence for the PP precursors from the discontinuities even in the presence of well-developed PP waveforms. Non-detections are found in six different data sets consisting of tens to hundreds of events. We use synthetic modelling to examine a suite of factors that could be responsible for the absence of the PP precursors. The take-off angles for PP and the precursors differ by only 1.2-1.5°; thus source-related complexity would affect PP and the precursors. A PP wave attenuated in the upper mantle would increase the relative amplitude of the PP precursors. Attenuation within the transition zone could reduce precursor amplitudes, but this would be a regional phenomenon restricted to particular source receiver geometries. We also find little evidence for deviations from the theoretical travel path of seismic rays expected for scattered arrivals. Factors that have a strong influence include the stacking procedures used in seismic array techniques in the presence of large, interfering phases, the presence of topography on the discontinuities on the order of tens of kilometres, and 3-D lateral heterogeneity in

Sequence similarities and evolutionary relationships of microbial, plant and animal alpha-amylases.

PubMed

Janecek, S

1994-09-01

Amino acid sequence comparison of 37 alpha-amylases from microbial, plant and animal sources was performed to identify their mutual sequence similarities in addition to the five already described conserved regions. These sequence regions were examined from structure/function and evolutionary perspectives. An unrooted evolutionary tree of alpha-amylases was constructed on a subset of 55 residues from the alignment of sequence similarities along with conserved regions. The most important new information extracted from the tree was as follows: (a) the close evolutionary relationship of Alteromonas haloplanctis alpha-amylase (thermolabile enzyme from an antarctic psychrotroph) with the already known group of homologous alpha-amylases from streptomycetes, Thermomonospora curvata, insects and mammals, and (b) the remarkable 40.1% identity between starch-saccharifying Bacillus subtilis alpha-amylase and the enzyme from the ruminal bacterium Butyrivibrio fibrisolvens, an alpha-amylase with an unusually large polypeptide chain (943 residues in the mature enzyme). Due to a very high degree of similarity, the whole amino acid sequences of three groups of alpha-amylases, namely (a) fungi and yeasts, (b) plants, and (c) A. haloplanctis, streptomycetes, T. curvata, insects and mammals, were aligned independently and their unrooted distance trees were calculated using these alignments. Possible rooting of the trees was also discussed. Based on the knowledge of the location of the five disulfide bonds in the structure of pig pancreatic alpha-amylase, the possible disulfide bridges were established for each of these groups of homologous alpha-amylases.

Tall oil precursors in three western pines: ponderosa, lodgepole, and limber pine

DOE Office of Scientific and Technical Information (OSTI.GOV)

Conner, A.H.; Diehl, M.A.; Rowe, J.W.

1980-01-01

The nonvolatile diethyl ether extracts (NVEE) from ponderosa, lodgepole, and limber pines were analyzed to determine the amounts and chemical composition of the tall oil precursors (resin acids, fatty acids, and nonsaponifiables) and turpentine precursors available from these species. The results showed that crude tall oil compositions would be approximately as follows (% resin acids, % fatty acids, % nonsaponifiables); ponderosa pine - sapwood (15, 75, 10), heartwood (78, 7, 15); lodgepole pine - sapwood (24, 57, 19), heartwood (51, 26, 23); limber pine - sapwood (10, 82, 8), heartwood (23, 60, 17). The larger nonsaponifiables content, as compared tomore » southern pines, is the major factor in explaining the greater difficulty in the distillative refining of tall oil from these western species. Eight resin acids were found in ponderosa and lodgepole pine: palustric, isopimaric, abietic, dehydroabietic, and neoabietic acids predominated. Seven resin acids were identified from limber pine: anticopalic, isopimaric, abietic, and dehydroabietic acids predominated. The free and esterfied fatty acids from these species contained predominantly oleic and linoleic acids. In addition limber pine contained major amounts of 5, 9, 12-octadecatrienoic acid. The nonsaponifiables contained mostly diterpenes and the sterols, sitosterol and campesterol. The major turpentine components were: ponderosa pine - ..beta..-pinene and 3-carene; lodgepole pine - ..beta..-phellandrene; and limber pine - 3-carene, ..beta..-phellandrene, ..cap alpha..-piene, and ..beta..-pinene.« less

New insights into the capacity of commercial wine yeasts to grow on sparkling wine media. Factor screening for improving wine yeast selection.

PubMed

Borrull, Anna; Poblet, Montse; Rozès, Nicolas

2015-06-01

During the production of sparkling wine, wine yeasts are subjected to many stress factors apart from ethanol, which lead to the need to achieve their acclimation in line with various industrial protocols. In the present work, 44 commercial wine Saccharomyces cerevisiae strains and one laboratory strain (BY4742) were firstly subjected to the influence of increasing concentrations of ethanol to cluster the yeasts using discriminant function analysis. Afterwards, non-inhibitory concentration (NIC) and minimum inhibitory concentration (MIC) were estimated, revealing some differences between 24 of these strains. Meanwhile, this study confirms the negative synergistic effect of low pH with ethanol on the maximum specific growth rate (μmax) and lag phase time. Moreover, a negative effect of increasing levels of glycerol in the growth medium was observed. Interestingly enough, an interactive positive effect was found between cysteine and medium-chain fatty acids (MCFA). While cysteine did not have a really significant effect in comparison to the control, it was able to restore the damage caused by MCFA, making the growth rate of cells recover and even reducing the formation of reactive oxygen species. Adequate culture aeration is also crucial for the composition of the cell fatty acid. The final results showed that few differences were observed between NIC and MIC estimations with respect to cells pre-cultured in the presence or absence of oxygen. Copyright © 2014 Elsevier Ltd. All rights reserved.

The TCP4 transcription factor of Arabidopsis blocks cell division in yeast at G1 {yields} S transition

DOE Office of Scientific and Technical Information (OSTI.GOV)

Aggarwal, Pooja; Padmanabhan, Bhavna; Bhat, Abhay

2011-07-01

Highlights: {yields} TCP4 is a class II TCP transcription factor, that represses cell division in Arabidopsis. {yields} TCP4 expression in yeast retards cell division by blocking G1 {yields} S transition. {yields} Genome-wide expression studies and Western analysis reveals stabilization of cell cycle inhibitor Sic1, as possible mechanism. -- Abstract: The TCP transcription factors control important aspects of plant development. Members of class I TCP proteins promote cell cycle by regulating genes directly involved in cell proliferation. In contrast, members of class II TCP proteins repress cell division. While it has been postulated that class II proteins induce differentiation signal, theirmore » exact role on cell cycle has not been studied. Here, we report that TCP4, a class II TCP protein from Arabidopsis that repress cell proliferation in developing leaves, inhibits cell division by blocking G1 {yields} S transition in budding yeast. Cells expressing TCP4 protein with increased transcriptional activity fail to progress beyond G1 phase. By analyzing global transcriptional status of these cells, we show that expression of a number of cell cycle genes is altered. The possible mechanism of G1 {yields} S arrest is discussed.« less

Simultaneous improvement in production of microalgal biodiesel and high-value alpha-linolenic acid by a single regulator acetylcholine.

PubMed

Parsaeimehr, Ali; Sun, Zhilan; Dou, Xiao; Chen, Yi-Feng

2015-01-01

Photoautotrophic microalgae are a promising avenue for sustained biodiesel production, but are compromised by low yields of biomass and lipids at present. We are developing a chemical approach to improve microalgal accumulation of feedstock lipids as well as high-value alpha-linolenic acid which in turn might provide a driving force for biodiesel production. We demonstrate the effectiveness of the small bioactive molecule "acetylcholine" on accumulation of biomass, total lipids, and alpha-linolenic acid in Chlorella sorokiniana. The effectiveness exists in different species of Chlorella. Moreover, the precursor and analogs of acetylcholine display increased effectiveness at higher applied doses, with maximal increases by 126, 80, and 60% over controls for biomass, total lipids, and alpha-linolenic acid, respectively. Production of calculated biodiesel was also improved by the precursor and analogs of acetylcholine. The biodiesel quality affected by changes in microalgal fatty acid composition was addressed. The chemical approach described here could improve the lipid yield and biodiesel production of photoautotrophic microalgae if combined with current genetic approaches.

Mitochondria inheritance is a key factor for tolerance to dehydration in wine yeast production.

PubMed

Picazo, C; Gamero-Sandemetrio, E; Orozco, H; Albertin, W; Marullo, P; Matallana, E; Aranda, A

2015-03-01

Mitochondria are the cell's powerhouse when organisms are grown in the presence of oxygen. They are also the source of reactive oxygen species that cause damage to the biochemical components of the cell and lead to cellular ageing and death. Under winemaking conditions, Saccharomyces yeasts exclusively have a fermentative metabolism due to the high sugar content of grape must. However, their production as an active dry yeast (ADY) form required aerobic propagation and a dehydration process. In these industrial steps, oxidative stress is particularly harmful for the cell. In this work, we analysed the impact of the mitochondrial genome on oxidative stress response, longevity and dehydration tolerance using the synthetic interspecific hybrids obtained between two S. cerevisiae and S. uvarum strains. The isogenic nature of nuclear DNA of such hybrids allowed us to analyse the impact of mitochondrial DNA for fermentative and oxidative stress conditions. Under grape must conditions, the inheritance of mitochondrial DNA poorly impacted the fermentative performance of interspecific hybrids, unlike the hybrids with S. cerevisiae mitochondrial inheritance, which displayed increased tolerance to oxidative stress and dehydration, and showed an extended chronological longevity when cells were grown with aeration. In modern oenology, yeast starters are employed to inoculate grape juice, usually in the form of active dry yeast (ADY). The dehydration process implies stressful conditions that lead to oxidative damage. Other yeast species and interspecific hybrids other than Saccharomyces cerevisiae may be used to confer novel properties to the final product. However, these yeasts are usually more sensitive to drying. Understanding the causes of oxidative stress tolerance is therefore necessary for developing the use of these organisms in industry. This study indicates the impact of mitochondrial DNA inheritance for oxidative stress resistance in an interspecific context using

Responses of Yeast Biocontrol Agents to Environmental Stress

PubMed Central

Sui, Yuan; Wisniewski, Michael; Droby, Samir

2015-01-01

Biological control of postharvest diseases, utilizing wild species and strains of antagonistic yeast species, is a research topic that has received considerable attention in the literature over the past 30 years. In principle, it represents a promising alternative to chemical fungicides for the management of postharvest decay of fruits, vegetables, and grains. A yeast-based biocontrol system is composed of a tritrophic interaction between a host (commodity), a pathogen, and a yeast species, all of which are affected by environmental factors such as temperature, pH, and UV light as well as osmotic and oxidative stresses. Additionally, during the production process, biocontrol agents encounter various severe abiotic stresses that also impact their viability. Therefore, understanding the ecological fitness of the potential yeast biocontrol agents and developing strategies to enhance their stress tolerance are essential to their efficacy and commercial application. The current review provides an overview of the responses of antagonistic yeast species to various environmental stresses, the methods that can be used to improve stress tolerance and efficacy, and the related mechanisms associated with improved stress tolerance. PMID:25710368

Regulation of hypoxia-inducible factor-1alpha by nitric oxide through mitochondria-dependent and -independent pathways.

PubMed Central

Mateo, Jesús; García-Lecea, Marta; Cadenas, Susana; Hernández, Carlos; Moncada, Salvador

2003-01-01

Nitric oxide (NO) has been reported both to promote and to inhibit the activity of the transcription factor hypoxia-inducible factor-1 (HIF-1). In order to avoid the pitfalls associated with the use of NO donors, we have developed a human cell line (Tet-iNOS 293) that expresses the inducible NO synthase (iNOS) under the control of a tetracycline-inducible promoter. Using this system to generate finely controlled amounts of NO, we have demonstrated that the stability of the alpha-subunit of HIF-1 is regulated by NO through two separate mechanisms, only one of which is dependent on a functional respiratory chain. HIF-1alpha is unstable in cells maintained at 21% O(2), but is progressively stabilized as the O(2) concentration decreases, resulting in augmented HIF-1 DNA-binding activity. High concentrations of NO (>1 microM) stabilize HIF-1alpha at all O(2) concentrations tested. This effect does not involve the respiratory chain, since it is preserved in cells lacking functional mitochondria (rho(0)-cells) and is not reproduced by other inhibitors of the cytochrome c oxidase. By contrast, lower concentrations of NO (<400 nM) cause a rapid decrease in HIF-1alpha stabilized by exposure of the cells to 3% O(2). This effect of NO is dependent on the inhibition of mitochondrial respiration, since it is mimicked by other inhibitors of mitochondrial respiration, including those not acting at cytochrome c oxidase. We suggest that, although stabilization of HIF-1alpha by high concentrations of NO might have implications in pathophysiological processes, the inhibitory effect of lower NO concentrations is likely to be of physiological relevance. PMID:14531732

High glucose concentrations attenuate hypoxia-inducible factor-1{alpha} expression and signaling in non-tumor cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dehne, Nathalie, E-mail: dehne@biochem.uni-frankfurt.de; Hintereder, Gudrun, E-mail: Gudrun.Hintereder@kgu.de; Bruene, Bernhard, E-mail: bruene@pathobiochemie1.de

2010-04-15

Hypoxia-inducible factor (HIF) is the major transcription factor mediating adaption to hypoxia e.g. by enhancing glycolysis. In tumor cells, high glucose concentrations are known to increase HIF-1{alpha} expression even under normoxia, presumably by enhancing the concentration of tricarboxylic acid cycle intermediates, while reactions of non-tumor cells are not well defined. Therefore, we analyzed cellular responses to different glucose concentrations in respect to HIF activation comparing tumor to non-tumor cells. Using cells derived from non-tumor origin, we show that HIF-1{alpha} accumulation was higher under low compared to high glucose concentrations. Low glucose allowed mRNA expression of HIF-1 target genes like adrenomedullin.more » Transfection of C{sub 2}C{sub 12} cells with a HIF-1{alpha} oxygen-dependent degradation domaine-GFP fusion protein revealed that prolyl hydroxylase (PHD) activity is impaired at low glucose concentrations, thus stabilizing the fusion protein. Mechanistic considerations suggested that neither O{sub 2} redistribution nor an altered redox state explains impaired PHD activity in the absence of glucose. In order to affect PHD activity, glucose needs to be metabolized. Amino acids present in the medium also diminished HIF-1{alpha} expression, while the addition of fatty acids did not. This suggests that glucose or amino acid metabolism increases oxoglutarate concentrations, which enhances PHD activity in non-tumor cells. Tumor cells deprived of glutamine showed HIF-1{alpha} accumulation in the absence of glucose, proposing that enhanced glutaminolysis observed in many tumors enables these cells to compensate reduced oxoglutarate production in the absence of glucose.« less

Cloning and purification of alpha-neurotoxins from king cobra (Ophiophagus hannah).

PubMed

He, Ying-Ying; Lee, Wei-Hui; Zhang, Yun

2004-09-01

Thirteen complete and three partial cDNA sequences were cloned from the constructed king cobra (Ophiophagus hannah) venom gland cDNA library. Phylogenetic analysis of nucleotide sequences of king cobra with those from other snake venoms revealed that obtained cDNAs are highly homologous to snake venom alpha-neurotoxins. Alignment of deduced mature peptide sequences of the obtained clones with those of other reported alpha-neurotoxins from the king cobra venom indicates that our obtained 16 clones belong to long-chain neurotoxins (seven), short-chain neurotoxins (seven), weak toxin (one) and variant (one), respectively. Up to now, two out of 16 newly cloned king cobra alpha-neurotoxins have identical amino acid sequences with CM-11 and Oh-6A/6B, which have been characterized from the same venom. Furthermore, five long-chain alpha-neurotoxins and two short-chain alpha-neurotoxins were purified from crude venom and their N-terminal amino acid sequences were determined. The cDNAs encoding the putative precursors of the purified native peptide were also determined based on the N-terminal amino acid sequencing. The purified alpha-neurotoxins showed different lethal activities on mice.

Alterations of hypoxia-inducible factor-1 alpha in the hippocampus of mice acutely and repeatedly exposed to hypoxia.

PubMed

Shao, Guo; Gao, Cui-Ying; Lu, Guo-Wei

2005-01-01

This work aims at investigating the effects of hypoxic preconditioning on hypoxia-inducible factor-1 alpha (HIF-1alpha) expression in the hippocampus of mice during acute and repeated hypoxic exposures. The mice were randomly divided into three groups and exposed, respectively, to hypoxia for 4 runs (group H4), 1 run (group H1), and 0 run (group H0). Reverse transcription-polymerase chain reaction (RT-PCR), Western blot, electrophoretic mobility shift assay (EMSA), and chromatin immunoprecipitation were used to examine the HIF-1alpha responses in the mouse hippocampus following exposure to hypoxia. The tolerance of mice to hypoxia increased significantly following acute and repetitive exposure to autoprogressive hypoxia. Total mRNA, total protein, and nuclear protein were extracted from the hippocampus for RT-PCR, Western blot, and EMSA, respectively. The HIF-1alpha mRNA levels were found to be increased in group H1 and decreased in group H4. The HIF-1alpha protein levels and HIF-1 DNA-binding activities were increased in group H1 and markedly increased in group H4. One of the HIF-1 target genes, vascular endothelial growth factor, increased in group H4. HIF-1 activation is thought to be involved in the protection of the brain of hypoxic preconditioned mice. Copyright 2005 S. Karger AG, Basel

Use of the Yeast Pichia pastoris as an Expression Host for Secretion of Enterocin L50, a Leaderless Two-Peptide (L50A and L50B) Bacteriocin from Enterococcus faecium L50▿

PubMed Central

Basanta, Antonio; Gómez-Sala, Beatriz; Sánchez, Jorge; Diep, Dzung B.; Herranz, Carmen; Hernández, Pablo E.; Cintas, Luis M.

2010-01-01

In this work, we report the expression and secretion of the leaderless two-peptide (EntL50A and EntL50B) bacteriocin enterocin L50 from Enterococcus faecium L50 by the methylotrophic yeast Pichia pastoris X-33. The bacteriocin structural genes entL50A and entL50B were fused to the Saccharomyces cerevisiae gene region encoding the mating pheromone α-factor 1 secretion signal (MFα1s) and cloned, separately and together (entL50AB), into the P. pastoris expression and secretion vector pPICZαA, which contains the methanol-inducible alcohol oxidase promoter (PAOX1) to express the fusion genes. After transfer into the yeast, the recombinant plasmids were integrated into the genome, resulting in three bacteriocinogenic yeast strains able to produce and secrete the individual bacteriocin peptides EntL50A and EntL50B separately and together. The secretion was efficiently directed by MFα1s through the Sec system, and the precursor peptides were found to be correctly processed to form mature and active bacteriocin peptides. The present work describes for the first time the heterologous expression and secretion of a two-peptide non-pediocin-like bacteriocin by a yeast. PMID:20348300

The Yeast Forkhead Transcription Factors Fkh1 and Fkh2 Regulate Lifespan and Stress Response Together with the Anaphase-Promoting Complex

PubMed Central

Postnikoff, Spike D. L.; Malo, Mackenzie E.; Wong, Berchman; Harkness, Troy A. A.

2012-01-01

Forkhead box O (FOXO) transcription factors have a conserved function in regulating metazoan lifespan. A key function in this process involves the regulation of the cell cycle and stress responses including free radical scavenging. We employed yeast chronological and replicative lifespan assays, as well as oxidative stress assays, to explore the potential evolutionary conservation of function between the FOXOs and the yeast forkhead box transcription factors FKH1 and FKH2. We report that the deletion of both FKH genes impedes normal lifespan and stress resistance, particularly in stationary phase cells, which are non-responsive to caloric restriction. Conversely, increased expression of the FKHs leads to extended lifespan and improved stress response. Here we show the Anaphase-Promoting Complex (APC) genetically interacts with the Fkh pathway, likely working in a linear pathway under normal conditions, as fkh1Δ fkh2Δ post-mitotic survival is epistatic to that observed in apc5CA mutants. However, under stress conditions, post-mitotic survival is dramatically impaired in apc5CA fkh1Δ fkh2Δ, while increased expression of either FKH rescues APC mutant growth defects. This study establishes the FKHs role as evolutionarily conserved regulators of lifespan in yeast and identifies the APC as a novel component of this mechanism under certain conditions, likely through combined regulation of stress response, genomic stability, and cell cycle regulation. PMID:22438832

The Influence of Heating Mains on Yeast Communities in Urban Soils

NASA Astrophysics Data System (ADS)

Tepeeva, A. N.; Glushakova, A. M.; Kachalkin, A. V.

2018-04-01

The number and species diversity of yeasts in urban soils (urbanozems) affected by heating mains and in epiphytic yeast complexes of grasses growing above them were studied. The number of yeasts in the soil reached 103-104 CFU/g; on the plants, 107 CFU/g. Significant (by an order of magnitude) increase in the total number of soil yeasts in the zone of heating mains in comparison with the surrounding soil was found in winter period. Overall, 25 species of yeasts were isolated in our study. Yeast community of studied urbanozems was dominated by the Candida sake, an eurybiont of the temperate zone and other natural ecotopes with relatively low temperatures, but its share was minimal in the zone of heating mains. In general, the structure of soil and epiphytic yeast complexes in the zones of heating mains differed from that in the surrounding area by higher species diversity and a lower share of pigmented species among the epiphytic yeasts. The study demonstrated that the number and species structure of soil yeast communities in urban soils change significantly under the influence of the temperature factor and acquire a mosaic distribution pattern.

Tumor necrosis factor-alpha inhibits insulin's stimulating effect on glucose uptake and endothelium-dependent vasodilation in humans.

PubMed

Rask-Madsen, Christian; Domínguez, Helena; Ihlemann, Nikolaj; Hermann, Thomas; Køber, Lars; Torp-Pedersen, Christian

2003-10-14

Inflammatory mechanisms could be involved in the pathogenesis of both insulin resistance and atherosclerosis. Therefore, we aimed at examining whether the proinflammatory cytokine tumor necrosis factor (TNF)-alpha inhibits insulin-stimulated glucose uptake and insulin-stimulated endothelial function in humans. Healthy, lean male volunteers were studied. On each study day, 3 acetylcholine (ACh) or sodium nitroprusside (SNP) dose-response studies were performed by infusion into the brachial artery. Before and during the last 2 dose-response studies, insulin and/or TNF-alpha were coinfused. During infusion of insulin alone for 20 minutes, forearm glucose uptake increased by 220+/-44%. This increase was completely inhibited during coinfusion of TNF-alpha (started 10 min before insulin) with a more pronounced inhibition of glucose extraction than of blood flow. Furthermore, TNF-alpha inhibited the ACh forearm blood flow response (P<0.001), and this inhibition was larger during insulin infusion (P=0.01) but not further increased by NG-monomethyl-L-arginine acetate (P=0.2). Insulin potentiated the SNP response less than the ACh response and the effect of TNF-alpha was smaller (P<0.001); TNF-alpha had no effect on the SNP response without insulin infusion. Thus, TNF-alpha inhibition of the combined response to insulin and ACh was likely mediated through inhibition of NO production. These results support the concept that TNF-alpha could play a role in the development of insulin resistance in humans, both in muscle and in vascular tissue.

Molecular cloning and characterization of beluga whale (Delphinapterus leucas) interleukin-1beta and tumor necrosis factor-alpha.

PubMed Central

Denis, F; Archambault, D

2001-01-01

Interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) are cytokines produced primarily by monocytes and macrophages with regulatory effects in inflammation and multiple aspects of the immune response. As yet, no molecular data have been reported for IL-1beta and TNF-alpha of the beluga whale. In this study, we cloned and determined the entire cDNA sequence encoding beluga whale IL-1beta and TNF-alpha. The genetic relationship of the cytokine sequences was then analyzed with those from several mammalian species, including the human and the pig. The homology of beluga whale IL-1beta nucleic acid and deduced amino acid sequences with those from these mammalian species ranged from 74.6 to 86.0% and 62.7 to 77.1%, respectively, whereas that of TNF-alpha varied from 79.3 to 90.8% and 75.3 to 87.7%, respectively. Phylogenetic analyses based on deduced amino acid sequences showed that the beluga whale IL-1beta and TNF-alpha were most closely related to those of the ruminant species (cattle, sheep, and deer). The beluga whale IL-1beta- and TNF-alpha-encoding sequences were thereafter successfully expressed in Escherichia coli as fusion proteins by using procaryotic expression vectors. The fusion proteins were used to produce beluga whale IL-1beta- and TNF-alpha-specific rabbit antisera. Images Figure 3. Figure 4. Figure 5. PMID:11768130

Mycophenolic acid attenuates tumor necrosis factor-alpha-induced endothelin-1 production in human aortic endothelial cells.

PubMed

Yang, Won Seok; Lee, Joo Mi; Han, Nam Jeong; Kim, Yoon Ji; Chang, Jai Won; Park, Su-Kil

2010-07-01

Atherosclerotic cardiovascular disease is the major cause of morbidity and mortality in solid organ transplant recipients. Endothelin-1 (ET-1) is implicated in the pathogenesis of atherosclerosis and is one of the potential therapeutic targets. This study was conducted to evaluate the effect of mycophenolic acid (MPA), an immunosuppressant for the transplant recipients, on tumor necrosis factor-alpha (TNF-alpha)-induced ET-1 production in aortic endothelial cells. In cultured human aortic endothelial cells, TNF-alpha increased ET-1 through AP-1 and NF-kappaB, whereas MPA attenuated it by reducing both AP-1 and NF-kappaB DNA-binding activities. TNF-alpha increased ET-1 via c-Jun NH2-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK), but not extracellular signal-regulated kinase. N-acetylcysteine that downregulated TNF-alpha-induced reactive oxygen species (ROS) inhibited JNK activation, but not p38 MAPK. N-acetylcysteine, SP600125 (JNK inhibitor) and SB203580 (p38 MAPK inhibitor) attenuated TNF-alpha-induced DNA-binding activities of both AP-1 and NF-kappaB. MPA inhibited JNK and p38 MAPK activations as well as ROS generation. N-acetylcysteine, SP600125, SB203580 and MPA had no effect on either TNF-alpha-induced IkappaBalpha degradation or p65 nuclear translocation, but attenuated p65 Ser276 phosphorylation. MPA attenuated TNF-alpha-induced ET-1 production through inhibitions of ROS-dependent JNK and ROS-independent p38 MAPK that regulated NF-kappaB as well as AP-1. These findings suggest that MPA could have an effect of amelioration of atherosclerosis. Copyright (c) 2010 Elsevier Ireland Ltd. All rights reserved.

Nitric oxide provokes tumor necrosis factor-alpha expression in adult feline myocardium through a cGMP-dependent pathway.

PubMed

Kalra, D; Baumgarten, G; Dibbs, Z; Seta, Y; Sivasubramanian, N; Mann, D L

2000-09-12

The mechanism(s) responsible for the persistent coexpression of tumor necrosis factor-alpha (TNF-alpha) and nitric oxide (NO) in the failing heart is unknown. To determine whether NO was sufficient to provoke TNF-alpha biosynthesis, we examined the effects of an NO donor, S-nitroso-N-acetyl penicillamine (SNAP), in buffer-perfused Langendorff hearts. SNAP (1 micromol/L) treatment resulted in a time- and dose-dependent increase in myocardial TNF-alpha mRNA and protein biosynthesis in adult cat hearts. The effects of SNAP were completely abrogated by a NO quenching agent, 2-(4-carboxyphenyl)-4, 4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (C-PTIO), and mimicked by sodium nitroprusside. Electrophoretic mobility shift assays demonstrated that SNAP treatment led to the rapid induction of nuclear factor kappa-beta (NF-kappaB) but not AP-1. The importance of the cGMP pathway in terms of mediating NO-induced TNF-alpha biosynthesis was shown by studies that demonstrated that 8-bromo-cGMP mimicked the effects of SNAP and that the effects of SNAP could be completely abrogated using a cGMP antagonist, 1H-(1,2, 4)oxadiazolo(4,3-a)quinoxalin-1-one (ODQ), or protein kinase G antagonist (Rp-8-Br-cGMPS). SNAP and 8-Br-cGMP were both sufficient to lead to the site-specific phosphorylation (serine 32) and degradation of IkappaBalpha in isolated cardiac myocytes. Finally, protein kinase G was sufficient to directly phosphorylate IkappaBalpha on serine 32, a critical step in the activation of NF-kappaB. These studies show that NO provokes TNF-alpha biosynthesis through a cGMP-dependent pathway, which suggests that the coincident expression of TNF-alpha and NO may foster self-sustaining positive autocrine/paracrine feedback inflammatory circuits within the failing heart.

Kinetics of growth and sugar consumption in yeasts.

PubMed

van Dijken, J P; Weusthuis, R A; Pronk, J T

1993-01-01

An overview is presented of the steady- and transient state kinetics of growth and formation of metabolic byproducts in yeasts. Saccharomyces cerevisiae is strongly inclined to perform alcoholic fermentation. Even under fully aerobic conditions, ethanol is produced by this yeast when sugars are present in excess. This so-called 'Crabtree effect' probably results from a multiplicity of factors, including the mode of sugar transport and the regulation of enzyme activities involved in respiration and alcoholic fermentation. The Crabtree effect in S. cerevisiae is not caused by an intrinsic inability to adjust its respiratory activity to high glycolytic fluxes. Under certain cultivation conditions, for example during growth in the presence of weak organic acids, very high respiration rates can be achieved by this yeast. S. cerevisiae is an exceptional yeast since, in contrast to most other species that are able to perform alcoholic fermentation, it can grow under strictly anaerobic conditions. 'Non-Saccharomyces' yeasts require a growth-limiting supply of oxygen (i.e. oxygen-limited growth conditions) to trigger alcoholic fermentation. However, complete absence of oxygen results in cessation of growth and therefore, ultimately, of alcoholic fermentation. Since it is very difficult to reproducibly achieve the right oxygen dosage in large-scale fermentations, non-Saccharomyces yeasts are therefore not suitable for large-scale alcoholic fermentation of sugar-containing waste streams. In these yeasts, alcoholic fermentation is also dependent on the type of sugar. For example, the facultatively fermentative yeast Candida utilis does not ferment maltose, not even under oxygen-limited growth conditions, although this disaccharide supports rapid oxidative growth.

Hepatocyte nuclear factor 4alpha contributes to thyroid hormone homeostasis by cooperatively regulating the type 1 iodothyronine deiodinase gene with GATA4 and Kruppel-like transcription factor 9.

PubMed

Ohguchi, Hiroto; Tanaka, Toshiya; Uchida, Aoi; Magoori, Kenta; Kudo, Hiromi; Kim, Insook; Daigo, Kenji; Sakakibara, Iori; Okamura, Masashi; Harigae, Hideo; Sasaki, Takeshi; Osborne, Timothy F; Gonzalez, Frank J; Hamakubo, Takao; Kodama, Tatsuhiko; Sakai, Juro

2008-06-01

Type 1 iodothyronine deiodinase (Dio1), a selenoenzyme catalyzing the bioactivation of thyroid hormone, is highly expressed in the liver. Dio1 mRNA and enzyme activity levels are markedly reduced in the livers of hepatocyte nuclear factor 4alpha (HNF4alpha)-null mice, thus accounting for its liver-specific expression. Consistent with this deficiency, serum T4 and rT3 concentrations are elevated in these mice compared with those in HNF4alpha-floxed control littermates; however, serum T3 levels are unchanged. Promoter analysis of the mouse Dio1 gene demonstrated that HNF4alpha plays a key role in the transactivation of the mouse Dio1 gene. Deletion and substitution mutation analyses demonstrated that a proximal HNF4alpha site (direct repeat 1 [TGGACAAAGGTGC]; HNF4alpha-RE) is crucial for transactivation of the mouse Dio1 gene by HNF4alpha. Mouse Dio1 is also stimulated by thyroid hormone signaling, but a direct role for thyroid hormone receptor action has not been reported. We also showed that thyroid hormone-inducible Krüppel-like factor 9 (KLF9) stimulates the mouse Dio1 promoter very efficiently through two CACCC sequences that are located on either side of HNF4alpha-RE. Furthermore, KLF9 functions together with HNF4alpha and GATA4 to synergistically activate the mouse Dio1 promoter, suggesting that Dio1 is regulated by thyroid hormone in the mouse through an indirect mechanism requiring prior KLF9 induction. In addition, we showed that physical interactions between the C-terminal zinc finger domain (Cf) of GATA4 and activation function 2 of HNF4alpha and between the basic domain adjacent to Cf of GATA4 and a C-terminal domain of KLF9 are both required for this synergistic response. Taken together, these results suggest that HNF4alpha regulates thyroid hormone homeostasis through transcriptional regulation of the mouse Dio1 gene with GATA4 and KLF9.

Arabidopsis thaliana alpha1,2-glucosyltransferase (ALG10) is required for efficient N-glycosylation and leaf growth

PubMed Central

Farid, Akhlaq; Pabst, Martin; Schoberer, Jennifer; Altmann, Friedrich; Glössl, Josef; Strasser, Richard

2011-01-01

Assembly of the dolichol-linked oligosaccharide precursor (Glc3Man9GlcNAc2) is highly conserved among eukaryotes. In contrast to yeast and mammals, little is known about the biosynthesis of dolichol-linked oligosaccharides and the transfer to asparagine residues of nascent polypeptides in plants. To understand the biological function of these processes in plants we characterized the Arabidopsis thaliana homolog of yeast ALG10, the α1,2-glucosyltransferase that transfers the terminal glucose residue to the lipid-linked precursor. Expression of an Arabidopsis ALG10–GFP fusion protein in Nicotiana benthamiana leaf epidermal cells revealed a reticular distribution pattern resembling endoplasmic reticulum (ER) localization. Analysis of lipid-linked oligosaccharides showed that Arabidopsis ALG10 can complement the yeast Δalg10 mutant strain. A homozygous Arabidopsis T-DNA insertion mutant (alg10-1) accumulated mainly lipid-linked Glc2Man9GlcNAc2 and displayed a severe protein underglycosylation defect. Phenotypic analysis of alg10-1 showed that mutant plants have altered leaf size when grown in soil. Moreover, the inactivation of ALG10 in Arabidopsis resulted in the activation of the unfolded protein response, increased salt sensitivity and suppression of the phenotype of α-glucosidase I-deficient plants. In summary, these data show that Arabidopsis ALG10 is an ER-resident α1,2-glucosyltransferase that is required for lipid-linked oligosaccharide biosynthesis and subsequently for normal leaf development and abiotic stress response. PMID:21707802

Functional properties of an isolated. cap alpha beta. heterodimeric human placenta insulin-like growth factor 1 receptor complex

DOE Office of Scientific and Technical Information (OSTI.GOV)

Feltz, S.M.; Swanson, M.L.; Wemmie, J.A.

1988-05-03

Treatment of human placenta membranes at pH 8.5 in the presence of 2.0 mM dithiothreitol (DTT) for 5 min, followed by the simultaneous removal of the DTT and pH adjustment of pH 7.6, resulted in the formation of a functional ..cap alpha beta.. heterodimeric insulin-like growth factor 1 (IGF-1) receptor complex from the native ..cap alpha../sub 2/..beta../sub 2/ heterotetrameric disulfide-linked state. The membrane-bound ..cap alpha beta.. heterodimeric complex displayed similar curvilinear /sup 125/I-IGF-1 equilibrium binding compared to the ..cap alpha../sub 2/..beta../sub 2/ heterotetrameric complex. /sup 125/I-IGF-1 binding to both the isolated ..cap alpha../sub 2/..beta../sub 2/ heterotetrameric and ..cap alpha beta..more » heterodimeric complexes demonstrated a marked straightening of the Scatchard plots, compared to the placenta membrane-bound IGF-1 receptors, with a 2-fold increase in the high-affinity binding component. IGF-1 stimulation of IGF-1 receptor autophosphorylation indicated that the ligand-dependent activation of ..cap alpha beta.. heterodimeric protein kinase activity occurred concomitant with the reassociation into a covalent ..cap alpha../sub 2/..beta../sub 2/ heterotetrameric state. These data demonstrate that (i) a combination of alkaline pH and DTT treatment of human placenta membranes results in the formation of an ..cap alpha beta.. heterodimeric IGF-1 receptor complex, (ii) unlike the insulin receptor, high-affinity homogeneous IGF-1 binding occurs in both the ..cap alpha../sub 2/..beta../sub 2/ heterotetrameric and ..cap alpha beta.. heterodimeric complexes, and (iii) IGF-1-dependent autophosphorylation of the ..cap alpha beta.. heterodimeric IGF-1 receptor complex correlates wit an IGF-1 dependent covalent reassociation into an ..cap alpha../sub 2/..beta../sub 2/ heterotetrameric disulfide-linked state.« less

Prions in Yeast

PubMed Central

Liebman, Susan W.; Chernoff, Yury O.

2012-01-01

The concept of a prion as an infectious self-propagating protein isoform was initially proposed to explain certain mammalian diseases. It is now clear that yeast also has heritable elements transmitted via protein. Indeed, the “protein only” model of prion transmission was first proven using a yeast prion. Typically, known prions are ordered cross-β aggregates (amyloids). Recently, there has been an explosion in the number of recognized prions in yeast. Yeast continues to lead the way in understanding cellular control of prion propagation, prion structure, mechanisms of de novo prion formation, specificity of prion transmission, and the biological roles of prions. This review summarizes what has been learned from yeast prions. PMID:22879407

Thioredoxin-independent regulation of metabolism by the alpha-arrestin proteins.

PubMed

Patwari, Parth; Chutkow, William A; Cummings, Kiersten; Verstraeten, Valerie L R M; Lammerding, Jan; Schreiter, Eric R; Lee, Richard T

2009-09-11

Thioredoxin-interacting protein (Txnip), originally characterized as an inhibitor of thioredoxin, is now known to be a critical regulator of glucose metabolism in vivo. Txnip is a member of the alpha-arrestin protein family; the alpha-arrestins are related to the classical beta-arrestins and visual arrestins. Txnip is the only alpha-arrestin known to bind thioredoxin, and it is not known whether the metabolic effects of Txnip are related to its ability to bind thioredoxin or related to conserved alpha-arrestin function. Here we show that wild type Txnip and Txnip C247S, a Txnip mutant that does not bind thioredoxin in vitro, both inhibit glucose uptake in mature adipocytes and in primary skin fibroblasts. Furthermore, we show that Txnip C247S does not bind thioredoxin in cells, using thiol alkylation to trap the Txnip-thioredoxin complex. Because Txnip function was independent of thioredoxin binding, we tested whether inhibition of glucose uptake was conserved in the related alpha-arrestins Arrdc4 and Arrdc3. Both Txnip and Arrdc4 inhibited glucose uptake and lactate output, while Arrdc3 had no effect. Structure-function analysis indicated that Txnip and Arrdc4 inhibit glucose uptake independent of the C-terminal WW-domain binding motifs, recently identified as important in yeast alpha-arrestins. Instead, regulation of glucose uptake was intrinsic to the arrestin domains themselves. These data demonstrate that Txnip regulates cellular metabolism independent of its binding to thioredoxin and reveal the arrestin domains as crucial structural elements in metabolic functions of alpha-arrestin proteins.

Tumor necrosis factor-{alpha} induces MMP-9 expression via p42/p44 MAPK, JNK, and nuclear factor-{kappa}B in A549 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lin, C.-C.; Tseng, Hsiao-Wei; Hsieh, Hsi-Lung

2008-06-15

Matrix metalloproteinases (MMPs), in particular MMP-9, have been shown to be induced by cytokines including tumor necrosis factor-{alpha} (TNF-{alpha}) and contributes to airway inflammation. However, the mechanisms underlying MMP-9 expression induced by TNF-{alpha} in human A549 cells remain unclear. Here, we showed that TNF-{alpha} induced production of MMP-9 protein and mRNA is determined by zymographic, Western blotting, RT-PCR and ELISA assay, which were attenuated by inhibitors of MEK1/2 (U0126), JNK (SP600125), and NF-{kappa}B (helenalin), and transfection with dominant negative mutants of ERK2 ({delta}ERK) and JNK ({delta}JNK), and siRNAs for MEK1, p42 and JNK2. TNF-{alpha}-stimulated phosphorylation of p42/p44 MAPK and JNKmore » were attenuated by pretreatment with the inhibitors U0126 and SP600125 or transfection with dominant negative mutants of {delta}ERK and {delta}JNK. Furthermore, the involvement of NF-{kappa}B in TNF-{alpha}-induced MMP-9 production was consistent with that TNF-{alpha}-stimulated degradation of I{kappa}B-{alpha} and translocation of NF-{kappa}B into the nucleus which were blocked by helenalin, but not by U0126 and SP600125, revealed by immunofluorescence staining. The regulation of MMP-9 gene transcription by MAPKs and NF-{kappa}B was further confirmed by gene luciferase activity assay. MMP-9 promoter activity was enhanced by TNF-{alpha} in A549 cells transfected with wild-type MMP-9-Luc, which was inhibited by helenalin, U0126, or SP600125. In contrast, TNF-{alpha}-stimulated MMP-9 luciferase activity was totally lost in cells transfected with mutant-NF-{kappa}B MMP-9-luc. Moreover, pretreatment with actinomycin D and cycloheximide attenuated TNF-{alpha}-induced MMP-9 expression. These results suggest that in A549 cells, phosphorylation of p42/p44 MAPK, JNK, and transactivation of NF-{kappa}B are essential for TNF-{alpha}-induced MMP-9 gene expression.« less

PPAR-alpha agonist treatment increases trefoil factor family-3 expression and attenuates apoptosis in the liver tissue of bile duct-ligated rats.

PubMed

Karakan, Tarkan; Kerem, Mustafa; Cindoruk, Mehmet; Engin, Doruk; Alper, Murat; Akın, Okan

2013-01-01

Peroxisome proliferators-activated receptor alpha activation modulates cholesterol metabolism and suppresses bile acid synthesis. The trefoil factor family comprises mucin-associated proteins that increase the viscosity of mucins and help protect epithelial linings from insults. We evaluated the effect of short-term administration of fenofibrate, a peroxisome proliferators activated receptor alpha agonist, on trefoil factor family-3 expression, degree of apoptosis, generation of free radicals, and levels of proinflammatory cytokines in the liver tissue of bile duct-ligated rats. Forty male Wistar rats were randomly divided into four groups: 1 = sham operated, 2 = bile duct ligation, 3 = bile duct-ligated + vehicle (gum Arabic), and 4 = bile duct-ligated + fenofibrate (100 mg/kg/day). All rats were sacrificed on the 7 th day after obtaining blood samples and liver tissue. Liver function tests, tumor necrosis factor-alpha and interleukin 1 beta in serum, and trefoil factor family-3 mRNA expression, degree of apoptosis (TUNEL) and tissue malondialdehyde (malondialdehyde, end-product of lipid peroxidation by reactive oxygen species) in liver tissue were evaluated. Fenofibrate administration significantly reduced serum total bilirubin, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, gamma-glutamyl transferase, and tumor necrosis factor-alpha and interleukin-1β levels. Apoptosis and malondialdehyde were significantly reduced in the fenofibrate group. Trefoil factor family-3 expression increased with fenofibrate treatment in bile duct-ligated rats. The peroxisome proliferators-activated receptor alpha agonist fenofibrate significantly increased trefoil factor family-3 expression and decreased apoptosis and lipid peroxidation in the liver and attenuated serum levels of proinflammatory cytokines in bile duct-ligated rats. Further studies are needed to determine the protective role of fenofibrate in human cholestatic disorders.

Independent and additive effects of glutamic acid and methionine on yeast longevity.

PubMed

Wu, Ziyun; Song, Lixia; Liu, Shao Quan; Huang, Dejian

2013-01-01

It is established that glucose restriction extends yeast chronological and replicative lifespan, but little is known about the influence of amino acids on yeast lifespan, although some amino acids were reported to delay aging in rodents. Here we show that amino acid composition greatly alters yeast chronological lifespan. We found that non-essential amino acids (to yeast) methionine and glutamic acid had the most significant impact on yeast chronological lifespan extension, restriction of methionine and/or increase of glutamic acid led to longevity that was not the result of low acetic acid production and acidification in aging media. Remarkably, low methionine, high glutamic acid and glucose restriction additively and independently extended yeast lifespan, which could not be further extended by buffering the medium (pH 6.0). Our preliminary findings using yeasts with gene deletion demonstrate that glutamic acid addition, methionine and glucose restriction prompt yeast longevity through distinct mechanisms. This study may help to fill a gap in yeast model for the fast developing view that nutrient balance is a critical factor to extend lifespan.

Independent and Additive Effects of Glutamic Acid and Methionine on Yeast Longevity

PubMed Central

Wu, Ziyun; Song, Lixia; Liu, Shao Quan; Huang, Dejian

2013-01-01

It is established that glucose restriction extends yeast chronological and replicative lifespan, but little is known about the influence of amino acids on yeast lifespan, although some amino acids were reported to delay aging in rodents. Here we show that amino acid composition greatly alters yeast chronological lifespan. We found that non-essential amino acids (to yeast) methionine and glutamic acid had the most significant impact on yeast chronological lifespan extension, restriction of methionine and/or increase of glutamic acid led to longevity that was not the result of low acetic acid production and acidification in aging media. Remarkably, low methionine, high glutamic acid and glucose restriction additively and independently extended yeast lifespan, which could not be further extended by buffering the medium (pH 6.0). Our preliminary findings using yeasts with gene deletion demonstrate that glutamic acid addition, methionine and glucose restriction prompt yeast longevity through distinct mechanisms. This study may help to fill a gap in yeast model for the fast developing view that nutrient balance is a critical factor to extend lifespan. PMID:24244480

Comparative analysis of cell wall surface glycan expression in Candida albicans and Saccharomyces cerevisiae yeasts by flow cytometry.

PubMed

Martínez-Esparza, M; Sarazin, A; Jouy, N; Poulain, D; Jouault, T

2006-07-31

The yeast Candida albicans is an opportunistic pathogen, part of the normal human microbial flora that causes infections in immunocompromised individuals with a high morbidity and mortality levels. Recognition of yeasts by host cells is based on components of the yeast cell wall, which are considered part of its virulence attributes. Cell wall glycans play an important role in the continuous interchange that regulates the balance between saprophytism and parasitism, and also between resistance and infection. Some of these molecular entities are expressed both by the pathogenic yeast C. albicans and by Saccharomyces cerevisiae, a related non-pathogenic yeast, involving similar molecular mechanisms and receptors for recognition. In this work we have exploited flow cytometry methods for probing surface glycans of the yeasts. We compared glycan expression by C. albicans and by S. cerevisiae, and studied the effect of culture conditions. Our results show that the expression levels of alpha- and beta-linked mannosides as well as beta-glucans can be successfully evaluated by flow cytometry methods using different antibodies independent of agglutination reactions. We also found that the surface expression pattern of beta-mannosides detected by monoclonal or polyclonal antibodies are differently modulated during the growth course. These data indicate that the yeast beta-mannosides exposed on mannoproteins and/or phospholipomannan are increased in stationary phase, whereas those linked to mannan are not affected by the yeast growth phase. The cytometric method described here represents a useful tool to investigate to what extent C. albicans is able to regulate its glycan surface expression and therefore modify its virulence properties.

Purification and characterization of an alpha-amylase of Pichia burtonii isolated from the traditional starter "murcha" in Nepal.

PubMed

Takeuchi, Akiko; Shimizu-Ibuka, Akiko; Nishiyama, Yoshitaka; Mura, Kiyoshi; Okada, Sanae; Tokue, Chiyoko; Arai, Soichi

2006-12-01

Among more than 20 yeast strains isolated from the traditional starter "murcha" in Nepal, we characterized a yeast that might be involved in saccharification. This strain, identified as Pichia burtonii, produced an extracellular amylolytic enzyme when cultured in the presence of starch in the medium. Since no amylase secreted by P. burtonii has yet been reported, we purified the enzyme and determined its N-terminal amino acid sequence. Together with the results of a hydrolyzing activity assay toward various substrates, it was found to be an alpha-amylase. The purified enzyme, named Pichia burtonii alpha-amylase (PBA), was a glycoprotein with an apparent molecular mass of 51 kDa. Enzyme activity was optimal at pH 5.0 at 40 degrees C. The enzyme retained 80% of its original activity after incubation under the optimal pH condition at 50 degrees C for 30 min. The activity was inhibited by metal ions such as Cd(2+), Cu(2+), Hg(2+), Al(3+), and Zn(2+).

Roles of the bacterial cell wall and capsule in induction of tumor necrosis factor alpha by type III group B streptococci.

PubMed Central

Vallejo, J G; Baker, C J; Edwards, M S

1996-01-01

Group B streptococci (GBS) are the major cause of sepsis and fatal shock in neonates in the United States. The precise role of tumor necrosis factor alpha (TNF-alpha) in the development of human GBS sepsis has not been defined; however, whole GBS have been shown to induce the production of this inflammatory cytokine. We sought to determine which bacterial cell wall components of GBS are responsible for triggering TNF-alpha production. Human cord blood monocytes were stimulated with encapsulated (COH1) or unencapsulated (COH1-13) whole type III GBS or with purified bacterial components, including type III capsular polysaccharide (III-PS), group B polysaccharide (GB-PS), lipoteichoic acid (LTA), or peptidoglycan (PG). Lipopolysaccharide from Escherichia coli served as a control. Supernatants were harvested at specific timed intervals, and TNF-alpha levels were measured by enzyme-linked immunosorbent assay. Monocytes exposed to COH1 and COH1-13 induced similar amounts of TNF-alpha. III-PS, GB-PS, LTA, and PG each induced TNF-alpha in a time- and concentration-dependent manner. However, TNF-alpha release was significantly greater after stimulation by the GB-PS or PG than after stimulation by III-PS or LTA (P < 0.05). Our findings indicate that GB-PS and PG are the bacterial cell wall components primarily evoking TNF-alpha release. These, alone or in concert with other factors, may be responsible for septic shock accompanying GBS sepsis. PMID:8945544

Mitogenic action of tumour necrosis factor-alpha and interleukin-8 on explants of human duodenal mucosa.

PubMed

Zachrisson, K; Neopikhanov, V; Wretlind, B; Uribe, A

2001-08-07

Our aim is to examine whether tumour necrosis factor-alpha (TNF-alpha) and interleukin affect the mitotic activity in explants of human duodenal mucosa and to estimate the release of cytokines from explants incubated with TNF-alpha. Biopsy specimens of normal duodenal mucosa were taken from 19 subjects that underwent upper endoscopy for investigation of dyspeptic symptoms or chronic gastrointestinal bleeding. The specimens were processed following guidelines for organ culture technique. Paired biopsy specimens from 12 subjects were cultured for 23 h to achieve steady state and thereafter the explants were incubated 25 h with 10(-13)-10(-9) M of TNF-alpha or IL-8. Mitoses were arrested in the metaphase by adding vincristine sulphate for the last three hours. The explants were then fixed and processed for microdissection. Fifteen crypts were microdissected and the total number of metaphases was determined using the whole crypt as reference volume. The number of metaphases per crypt was also estimated in explants incubated with 10(-10) M TNF-alpha in the presence of anti-IL-8 antibodies. Additional duodenal explants from seven subjects were incubated with 10(-10) M TNF-alpha for 25 h. Thereafter the release of IL-1-beta, IL-6, IL-8 and interferon gamma (IFN-gamma) into the culture medium was measured by enzyme immunoassay and expressed as pg/mg protein. TNF-alpha and IL-8 significantly increased the number of metaphases/crypts (P<0.0001). The addition of anti-IL-8 slightly reduced the number of metaphases/crypt compared to the values observed in the explants incubated with 10(-10) M TNF-alpha alone (P<0.0001). The number of metaphases/crypt in the explants incubated with 10(-10) M TNF-alpha in the presence of anti-IL-8 antibodies was, however, markedly and significantly higher than that of the controls (P<0.000). TNF-alpha induced the release of IL-8 (P<0.01) and IL-6 (P<0.05) from the duodenal explants. TNF-alpha and IL-8 are potent mitogens to human small intestinal

[Effect of Xinmailong on hypoxia-inducible factor-1alpha expression in neonatal rats with asphyxia].

PubMed

Huang, Li-Xin; Wu, Xing-Heng

2009-08-01

Xinmailong, a compound extracted from Periplaneta americana, is used for the treatment of cardiovascular diseases. This study investigated the effects of Xinmailong on myocardial hypoxia-inducible factor-1alpha (HIF-1alpha) and plasma endothelin-1(ET-1) levels in neonatal rats with asphyxia and explored the protection mechanism of Xinmailong in hypoxia-ischemic myocardial injury. Seven-day-old Sprague-Dawley rats were randomly divided into three groups (n=30 each): sham-operated, asphyxia, Xinmailong-treated asphyxia. Each group was randomly subdivided into three groups according to the observed time points: 6 hrs, 24 hrs and 72 hrs. Xinmailong (5 mg/kg) was intraperitoneally injected to the rats in the Xinmailong-treated group five minutes before asphyxia. Myocardial HIF-1alpha expression, and plasma ET-1 and creatine kinase (CK) levels were measured. The histopathologic changes of the myocardium were observed by hematoxylin-eosin staining. Four rats died in the asphyxia group while only one died in the Xinmailong-treated group during the experiment. The plasma ET-1 and CK levels as well as myocardial HIF-1alpha expression increased at 6 hrs, reached a peak at 24 hrs, and declined at 72 hrs after asphyxia in the asphyxia group, being higher than that in the sham-operated group (P<0.01). Myocardial ischemia was observed in the three time points, and cell necrosis occurred at 24 hrs after asphyxia in the asphyxia group. Myocardial HIF-1alpha expression was positively correlated with plasma ET-1 levels (r=0.876, P<0.01). In the Xinmailong-treated group, plasma levels of CK and ET-1 as well as myocardial HIF-1alpha expression were significantly lower than those in the asphyxia group (P<0.01). Myocardial ischemia was alleviated and no cell necrosis was found in the Xinmailong-treated group. Asphyxia leads to increase in myocardial HIF-1alpha expression and plasma levels of ET-1 and CK. Xinmailong can reduce the myocardial expression of HIF-1alpha and decrease plasma ET

Human brain somatostatin release from isolated cortical nerve endings and its modulation through GABAB receptors.

PubMed Central

Bonanno, G.; Gemignani, A.; Schmid, G.; Severi, P.; Cavazzani, P.; Raiteri, M.

1996-01-01

1. The release of somatostatin-like immunoreactivity (SRIF-LI) in the human brain was studied in synaptosomal preparations from fresh neocortical specimens obtained from patients undergoing neurosurgery to remove deeply sited tumours. 2. The basal outflow of SRIF-LI from superfused synaptosomes was increased about 3 fold during exposure to a depolarizing medium containing 15 mM KCl. The K(+)-evoked overflow of SRIF-LI was almost totally dependent on the presence of Ca2+ in the superfusion medium. 3. The GABAB receptor agonist, (-)-baclofen (0.3 - 100 microM), inhibited the overflow of SRIF-LI in a concentration-dependent manner (EC50 = 1.84 +/- 0.20 microM; maximal effect: about 50%). The novel GABAB receptor ligand, 3-aminopropyl(difluoromethyl)phosphinic acid (CGP 47656) mimicked (-)-baclofen in inhibiting the SRIF-LI overflow (EC50 = 3.06 +/- 0.52 microM; maximal effect: about 50%), whereas the GABAA receptor agonist, muscimol, was ineffective up to 100 microM. 4. The inhibition by 10 microM (-)-baclofen of the K(+)-evoked SRIF-LI overflow was concentration-dependently prevented by two selective GABAB receptor antagonists, 3-amino-propyl (diethoxymethyl)-phosphinic acid (CGP 35348) (IC50 = 24.40 +/- 2.52 microM) and [3-[[(3,4-dichlorophenyl) methyl]amino]propyl] (diethoxymethyl) phosphinic acid (CGP 52432) (IC50 = 0.06 +/- 0.005 microM). 5. The inhibition of SRIF-LI overflow caused by 10 microM CGP 47656 was abolished by 1 microM CGP 52432. 6. When human synaptosomes were labelled with [3H]-GABA and depolarized in superfusion with 15 mM KCl, the inhibition by 10 microM (-)-baclofen of the depolarization-evoked [3H]-GABA overflow was largely prevented by 10 microM CGP 47656 which therefore behaved as an autoreceptor antagonist. 7. In conclusion: (a) the characteristics of SRIF-LI release from synaptosomal preparations of human neocortex are compatible with a neuronal origin; (b) the nerve terminals releasing the neuropeptide possess inhibitory receptors of the

A yeast-based genetic screening to identify human proteins that increase homologous recombination.

PubMed

Collavoli, Anita; Comelli, Laura; Rainaldi, Giuseppe; Galli, Alvaro

2008-05-01

To identify new human proteins implicated in homologous recombination (HR), we set up 'a papillae assay' to screen a human cDNA library using the RS112 strain of Saccharomyces cerevisiae containing an intrachromosomal recombination substrate. We isolated 23 cDNAs, 11 coding for complete proteins and 12 for partially deleted proteins that increased HR when overexpressed in yeast. We characterized the effect induced by the overexpression of the complete human proteasome subunit beta 2, the partially deleted proteasome subunits alpha 3 and beta 8, the ribosomal protein L12, the brain abundant membrane signal protein (BASP1) and the human homologue to v-Ha-RAS (HRAS), which elevated HR by 2-6.5-fold over the control. We found that deletion of the RAD52 gene, which has a key role in most HR events, abolished the increase of HR induced by the proteasome subunits and HRAS; by contrast, the RAD52 deletion did not affect the high level of HR due to BASP1 and RPL12. This suggests that the proteins stimulated yeast HR via different mechanisms. Overexpression of the complete beta 2 human proteasome subunit or the partially deleted alpha 3 and beta 8 subunits increased methyl methanesulphonate (MMS) resistance much more in the rad52 Delta mutant than in the wild-type. Overexpression of RPL12 and BASP1 did not affect MMS resistance in both the wild-type and the rad52 Delta mutant, whereas HRAS decreased MMS resistance in the rad52 Delta mutant. The results indicate that these proteins may interfere with the pathway(s) involved in the repair of MMS-induced DNA damage. Finally, we provide further evidence that yeast is a helpful tool to identify human proteins that may have a regulatory role in HR.

THE LYMAN ALPHA REFERENCE SAMPLE: EXTENDED LYMAN ALPHA HALOS PRODUCED AT LOW DUST CONTENT

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hayes, Matthew; Oestlin, Goeran; Duval, Florent

2013-03-10

We report on new imaging observations of the Lyman alpha emission line (Ly{alpha}), performed with the Hubble Space Telescope, that comprise the backbone of the Lyman alpha Reference Sample. We present images of 14 starburst galaxies at redshifts 0.028 < z < 0.18 in continuum-subtracted Ly{alpha}, H{alpha}, and the far ultraviolet continuum. We show that Ly{alpha} is emitted on scales that systematically exceed those of the massive stellar population and recombination nebulae: as measured by the Petrosian 20% radius, R{sub P20}, Ly{alpha} radii are larger than those of H{alpha} by factors ranging from 1 to 3.6, with an average ofmore » 2.4. The average ratio of Ly{alpha}-to-FUV radii is 2.9. This suggests that much of the Ly{alpha} light is pushed to large radii by resonance scattering. Defining the Relative Petrosian Extension of Ly{alpha} compared to H{alpha}, {xi}{sub Ly{alpha}} = R {sup Ly{alpha}}{sub P20}/R {sup H{alpha}}{sub P20}, we find {xi}{sub Ly{alpha}} to be uncorrelated with total Ly{alpha} luminosity. However, {xi}{sub Ly{alpha}} is strongly correlated with quantities that scale with dust content, in the sense that a low dust abundance is a necessary requirement (although not the only one) in order to spread Ly{alpha} photons throughout the interstellar medium and drive a large extended Ly{alpha} halo.« less

Elevation of CSF tumor necrosis factor alpha levels in new daily persistent headache and treatment refractory chronic migraine.

PubMed

Rozen, Todd; Swidan, Sahar Z

2007-01-01

To determine if patients with new daily persistent headache (NDPH) have elevated levels of tumor necrosis factor alpha (TNF alpha) in the CSF. NDPH is considered one of the most treatment resistant of all headache syndromes. This reflects a lack of understanding of its pathogenesis. As a certain percentage of NDPH patients have their headaches start after an infection, the possibility of a persistent state of systemic or CNS inflammation comes into question. TNF alpha is a proinflammatory cytokine involved in brain immune and inflammatory activities, as well as in pain initiation. The goal of this study was to look at TNF alpha levels in the CSF of NDPH patients, to determine if CNS inflammation may play some role in the pathogenesis of this condition. CSF TNF alpha levels were studied in 38 patients: 20 with NDPH and a control population of 16 patients with chronic migraine (CM), and 2 with post-traumatic headache (PT). CSF TNF alpha levels were elevated in 19 of 20 NDPH patients, 16 of 16 CM patients, and both PT patients. Serum TNF alpha levels were normal in most of the study subjects. An elevation of CSF TNF alpha levels was found in almost all NDPH patients and suggest a role for TNF alpha in the pathogenesis of this condition. Surprisingly, all CM and PT patients tested had elevated CSF TNF alpha levels. In most patients with elevated CSF levels, serum TNF alpha levels were normal. All of these syndromes may be manifestations of CNS inflammation. As most of the positive-tested patients showed minimal to no improvement during aggressive inpatient treatment, persistent elevation of CSF TNF alpha levels may be one of the causes of treatment refractory CDH.

Silsesquioxanes as precursors to ceramic composites

NASA Technical Reports Server (NTRS)

Hurwitz, Frances I.; Hyatt, Lizbeth H.; Gorecki, Joy; Damore, Lisa

1987-01-01

Silsesquioxanes having the general structure RSiO sub 1.5, where R = methyl, propyl, or phenyl, melt flow at 70 to 100 C. Above 100 C, free -OH groups condense. At 225 C further crosslinking occurs, and the materials form thermosets. Pyrolysis, with accompanying loss of volatiles, takes place at nominally 525 C. At higher temperatures, the R group serves as an internal carbon soruce for carbo-thermal reduction to SiC accompanied by the evolution of CO. By blending silsesquioxanes with varying R groups, both the melt rheology and composition of the fired ceramic can be controlled. Fibers can be spun from the melt which are stable in argon in 1400 C. The silsesquioxanes also were used as matrix precursors for Nicalon and alpha-SiC platelet reinforced composites.

Multiple roles of tumor necrosis factor-alpha in fracture healing.

PubMed

Karnes, Jonathan M; Daffner, Scott D; Watkins, Colleen M

2015-09-01

This review presents a summary of basic science evidence examining the influence of tumor necrosis factor-alpha (TNF-α) on secondary fracture healing. Multiple studies suggest that TNF-α, in combination with the host reservoir of peri-fracture mesenchymal stem cells, is a main determinant in the success of bone healing. Disease states associated with poor bone healing commonly have inappropriate TNF-α responses, which likely contributes to the higher incidence of delayed and nonunions in these patient populations. Appreciation of TNF-α in fracture healing may lead to new therapies to augment recovery and reduce the incidence of complications. Copyright © 2015 Elsevier Inc. All rights reserved.

Brain-Derived Neurotrophic Factor Induces Cell Survival and the Migration of Murine Adult Hippocampal Precursor Cells During Differentiation In Vitro.

PubMed

Ortiz-López, Leonardo; Vega-Rivera, Nelly Maritza; Babu, Harish; Ramírez-Rodríguez, Gerardo Bernabé

2017-01-01

The generation of new neurons during adulthood involves local precursor cell migration and terminal differentiation in the dentate gyrus. These events are influenced by the hippocampal microenvironment. Brain-derived neurotrophic factor (BDNF) is relevant for hippocampal neuronal development and behavior. Interestingly, studies that have been performed in controlled in vitro systems that involve isolated precursor cells that were derived from the dentate gyrus (AHPCs) have shown that BDNF induces the activation of the TrkB receptor and, consequentially, might activate signaling pathways that favor survival and neuronal differentiation. Based on the fact that the cellular events of AHPCs that are induced by single factors can be studied in this controlled in vitro system, we investigated the ability of BDNF and the involvement of protein kinase C (PKC), as one of the TrkB-downstream activated signaling proteins, in the regulation of migration, here reflected by motility, of AHPCs. Precursor cells were cultured following a concentration-response curve (1-640 ng/ml) for 24 or 96 h. We found that BDNF favored cell survival without altering the viability under culture proliferative conditions of the AHPCs. Concomitantly, glial- and neuronal-differentiated precursor cells increased as a consequence of survival promoted by BDNF. Additionally, pharmacological approaches showed that BDNF (40 ng/ml)-induced migration of AHPCs was blocked with the compounds K252a and GF109203x, which prevent the activation of TrkB and PKC, respectively. The results indicate that in the in vitro migration of differentiated AHPCs it is involved the BDNF and TrkB cascade. Our results provide additional information about the mechanism by which BDNF impacts adult neurogenesis in the hippocampus.

Yeast for virus research

PubMed Central

Zhao, Richard Yuqi

2017-01-01

Budding yeast (Saccharomyces cerevisiae) and fission yeast (Schizosaccharomyces pombe) are two popular model organisms for virus research. They are natural hosts for viruses as they carry their own indigenous viruses. Both yeasts have been used for studies of plant, animal and human viruses. Many positive sense (+) RNA viruses and some DNA viruses replicate with various levels in yeasts, thus allowing study of those viral activities during viral life cycle. Yeasts are single cell eukaryotic organisms. Hence, many of the fundamental cellular functions such as cell cycle regulation or programed cell death are highly conserved from yeasts to higher eukaryotes. Therefore, they are particularly suited to study the impact of those viral activities on related cellular activities during virus-host interactions. Yeasts present many unique advantages in virus research over high eukaryotes. Yeast cells are easy to maintain in the laboratory with relative short doubling time. They are non-biohazardous, genetically amendable with small genomes that permit genome-wide analysis of virologic and cellular functions. In this review, similarities and differences of these two yeasts are described. Studies of virologic activities such as viral translation, viral replication and genome-wide study of virus-cell interactions in yeasts are highlighted. Impacts of viral proteins on basic cellular functions such as cell cycle regulation and programed cell death are discussed. Potential applications of using yeasts as hosts to carry out functional analysis of small viral genome and to develop high throughput drug screening platform for the discovery of antiviral drugs are presented. PMID:29082230

Matrix factorization-based data fusion for gene function prediction in baker's yeast and slime mold.

PubMed

Zitnik, Marinka; Zupan, Blaž

2014-01-01

The development of effective methods for the characterization of gene functions that are able to combine diverse data sources in a sound and easily-extendible way is an important goal in computational biology. We have previously developed a general matrix factorization-based data fusion approach for gene function prediction. In this manuscript, we show that this data fusion approach can be applied to gene function prediction and that it can fuse various heterogeneous data sources, such as gene expression profiles, known protein annotations, interaction and literature data. The fusion is achieved by simultaneous matrix tri-factorization that shares matrix factors between sources. We demonstrate the effectiveness of the approach by evaluating its performance on predicting ontological annotations in slime mold D. discoideum and on recognizing proteins of baker's yeast S. cerevisiae that participate in the ribosome or are located in the cell membrane. Our approach achieves predictive performance comparable to that of the state-of-the-art kernel-based data fusion, but requires fewer data preprocessing steps.

Engineering of a mammalian O-glycosylation pathway in the yeast Saccharomyces cerevisiae: production of O-fucosylated epidermal growth factor domains.

PubMed

Chigira, Yuko; Oka, Takuji; Okajima, Tetsuya; Jigami, Yoshifumi

2008-04-01

Development of a heterologous system for the production of homogeneous sugar structures has the potential to elucidate structure-function relationships of glycoproteins. In the current study, we used an artificial O-glycosylation pathway to produce an O-fucosylated epidermal growth factor (EGF) domain in Saccharomyces cerevisiae. The in vivo O-fucosylation system was constructed via expression of genes that encode protein O-fucosyltransferase 1 and the EGF domain, along with genes whose protein products convert cytoplasmic GDP-mannose to GDP-fucose. This system allowed identification of an endogenous ability of S. cerevisiae to transport GDP-fucose. Moreover, expression of EGF domain mutants in this system revealed the different contribution of three disulfide bonds to in vivo O-fucosylation. In addition, lectin blotting revealed differences in the ability of fucose-specific lectin to bind the O-fucosylated structure of EGF domains from human factors VII and IX. Further introduction of the human fringe gene into yeast equipped with the in vivo O-fucosylation system facilitated the addition of N-acetylglucosamine to the EGF domain from factor IX but not from factor VII. The results suggest that engineering of an O-fucosylation system in yeast provides a powerful tool for producing proteins with homogenous carbohydrate chains. Such proteins can be used for the analysis of substrate specificity and the production of antibodies that recognize O-glycosylated EGF domains.

Modelling oxygen transfer using dynamic alpha factors.

PubMed

Jiang, Lu-Man; Garrido-Baserba, Manel; Nolasco, Daniel; Al-Omari, Ahmed; DeClippeleir, Haydee; Murthy, Sudhir; Rosso, Diego

2017-11-01

Due to the importance of wastewater aeration in meeting treatment requirements and due to its elevated energy intensity, it is important to describe the real nature of an aeration system to improve design and specification, performance prediction, energy consumption, and process sustainability. Because organic loadings drive aeration efficiency to its lowest value when the oxygen demand (energy) is the highest, the implications of considering their dynamic nature on energy costs are of utmost importance. A dynamic model aimed at identifying conservation opportunities is presented. The model developed describes the correlation between the COD concentration and the α factor in activated sludge. Using the proposed model, the aeration efficiency is calculated as a function of the organic loading (i.e. COD). This results in predictions of oxygen transfer values that are more realistic than the traditional method of assuming constant α values. The model was applied to two water resource recovery facilities, and was calibrated and validated with time-sensitive databases. Our improved aeration model structure increases the quality of prediction of field data through the recognition of the dynamic nature of the alpha factor (α) as a function of the applied oxygen demand. For the cases presented herein, the model prediction of airflow improved by 20-35% when dynamic α is used. The proposed model offers a quantitative tool for the prediction of energy demand and for minimizing aeration design uncertainty. Copyright © 2017 Elsevier Ltd. All rights reserved.

Novel, potent, and radio-iodinatable somatostatin receptor 1 (sst1) selective analogues.

PubMed

Erchegyi, Judit; Cescato, Renzo; Grace, Christy Rani R; Waser, Beatrice; Piccand, Véronique; Hoyer, Daniel; Riek, Roland; Rivier, Jean E; Reubi, Jean Claude

2009-05-14

The proposed sst(1) pharmacophore (J. Med. Chem. 2005, 48, 523-533) derived from the NMR structures of a family of mono- and dicyclic undecamers was used to design octa-, hepta-, and hexamers with high affinity and selectivity for the somatostatin sst(1) receptor. These compounds were tested for their in vitro binding properties to all five somatostatin (SRIF) receptors using receptor autoradiography; those with high SRIF receptor subtype 1 (sst(1)) affinity and selectivity were shown to be agonists when tested functionally in a luciferase reporter gene assay. Des-AA(1,4-6,10,12,13)-[DTyr(2),DAgl(NMe,2naphthoyl)(8),IAmp(9)]-SRIF-Thr-NH(2) (25) was radio-iodinated ((125)I-25) and specifically labeled sst(1)-expressing cells and tissues. 3D NMR structures were calculated for des-AA(1,4-6,10,12,13)-[DPhe(2),DTrp(8),IAmp(9)]-SRIF-Thr-NH(2) (16), des-AA(1,2,4-6,10,12,13)-[DAgl(NMe,2naphthoyl)(8),IAmp(9)]-SRIF-Thr-NH(2) (23), and des-AA(1,2,4-6,10,12,13)-[DAgl(NMe,2naphthoyl)(8),IAmp(9),Tyr(11)]-SRIF-NH(2) (27) in DMSO. Though the analogues have the sst(1) pharmacophore residues at the previously determined distances from each other, the positioning of the aromatic residues in 16, 23, and 27 is different from that described earlier, suggesting an induced fit mechanism for sst(1) binding of these novel, less constrained sst(1)-selective family members.

Interleukin-10 and tumour necrosis factor-alpha serum levels in chronic Chagas disease patients.

PubMed

Vasconcelos, R H T; Azevedo, E de A N; Diniz, G T N; Cavalcanti, M da G A de M; de Oliveira, W; de Morais, C N L; Gomes, Y de M

2015-07-01

In Chagas disease, chronically infected individuals may be asymptomatic or may present cardiac or digestive complications, and it is well known that the human immune response is related to different clinical manifestations. Different patterns of cytokine levels have been previously described in different clinical forms of this disease, but contradictory results are reported. Our aim was to evaluate the serum levels of interleukin-10 and tumour necrosis factor-alpha in patients with asymptomatic and cardiac Chagas disease. The serum interleukin-10 levels in patients with cardiomyopathy were higher than those in asymptomatic patients, mainly in those without heart enlargement. Although no significant difference was observed in serum tumour necrosis factor-alpha levels among the patients, we found that cardiac patients also present high levels of this cytokine, largely those with heart dilatation. Therefore, these cytokines play an important role in chronic Chagas disease cardiomyopathy. Follow-up investigations of these and other cytokines in patients with chronic Chagas disease need to be conducted to improve the understanding of the immunopathology of this disease. © 2015 John Wiley & Sons Ltd.

Nonequilibrium Green's functions theory for the alpha factor of quantum cascade lasers (Conference Presentation)

NASA Astrophysics Data System (ADS)

Pereira, Mauro F.; Winge, David O.; Wacker, Andreas; Jumpertz, Louise; Michel, Florian; Pawlus, Robert; Elsaesser, Wolfgang E.; Schires, Kevin; Carras, Mathieu; Grillot, Frédéric

2016-10-01

The linewidth of a conventional laser is due to fluctuations in the laser field due to spontaneous emission and described by the Schalow-Townes formula. In addition to that, in a semiconductor laser there is a contribution arising from fluctuations in the refractive index induced by carrier density fluctuations. The later are quantitatively described by the linewidth enhancement or alpha factor [C. H. Henry, IEEE J. Quantum Electron. 18 (2), 259 (1982), W. W. Chow, S. W. Koch and M. Sargent III, Semiconductor-Laser Physics, Springer-Verlag (1994), M.F. Pereira Jr et al, J. Opt. Soc. Am. B10, 765 (1993). In this paper we investigate the alpha factor of quantum cascade lasers under actual operating conditions using the Nonequilibrium Greens Functions approach [A. Wacker et a, IEEE Journal of Sel. Top. in Quantum Electron.,19 1200611, (2013), T. Schmielau and M.F. Pereira, Appl. Phys. Lett. 95 231111, (2009)]. The simulations are compared with recent results obtained with different optical feedback techniques [L. Jumpertz et al, AIP ADVANCES 6, 015212 (2016)].

L-arabinose fermenting yeast

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Min; Singh, Arjun; Suominen, Pirkko

An L-arabinose utilizing yeast strain is provided for the production of ethanol by introducing and expressing bacterial araA, araB and araD genes. L-arabinose transporters are also introduced into the yeast to enhance the uptake of arabinose. The yeast carries additional genomic mutations enabling it to consume L-arabinose, even as the only carbon source, and to produce ethanol. A yeast strain engineered to metabolize arabinose through a novel pathway is also disclosed. Methods of producing ethanol include utilizing these modified yeast strains.

L-arabinose fermenting yeast

DOEpatents

Zhang, Min; Singh, Arjun; Suominen, Pirkko; Knoshaug, Eric; Franden, Mary Ann; Jarvis, Eric

2014-09-23

An L-arabinose utilizing yeast strain is provided for the production of ethanol by introducing and expressing bacterial araA, araB and araD genes. L-arabinose transporters are also introduced into the yeast to enhance the uptake of arabinose. The yeast carries additional genomic mutations enabling it to consume L-arabinose, even as the only carbon source, and to produce ethanol. A yeast strain engineered to metabolize arabinose through a novel pathway is also disclosed. Methods of producing ethanol include utilizing these modified yeast strains.