scholarly journals Morphological and Biochemical Characterization of Macrophages Activated by Carrageenan and Lipopolysaccharide In Vivo

2004 ◽  
Vol 29 (2) ◽  
pp. 27-34 ◽  
Author(s):  
Valéria Pereira Nacife ◽  
Maria de Nazaré Correia Soeiro ◽  
Rachel Novaes Gomes ◽  
Heloísa D’Avila ◽  
Hugo Caire Castro-Faria Neto ◽  
...  
Keyword(s):  
Biochemical Characterization

Biochemical characterization of Yin Yang 1 – RNA complexes

2008 ◽  
Vol 86 (1) ◽  
pp. 31-36 ◽  
Author(s):  
Zachery R. Belak ◽  
Andrew Ficzycz ◽  
Nick Ovsenek
Keyword(s):  
Rna Binding ◽  
Biochemical Characterization ◽  
Ribonucleoprotein Complexes ◽  
Yin Yang 1 ◽  
Significant Difference ◽  
Yin Yang ◽  
Rna Interaction ◽  
Rna Complexes

YY1 (Yin Yang 1) is present in the Xenopus oocyte cytoplasm as a constituent of messenger ribonucleoprotein complexes (mRNPs). Association of YY1 with mRNPs requires direct RNA-binding activity. Previously, we have shown YY1 has a high affinity for U-rich RNA; however, potential interactions with plausible in vivo targets have not been investigated. Here we report a biochemical characterization of the YY1–RNA interaction including an investigation of the stability, potential 5′-methylguanosine affinity, and specificity for target RNAs. The formation of YY1–RNA complexes in vitro was highly resistant to thermal, ionic, and detergent disruption. The endogenous oocyte YY1–mRNA interactions were also found to be highly stable. Specific YY1–RNA interactions were observed with selected mRNA and 5S RNA probes. The affinity of YY1 for these substrates was within an order of magnitude of that for its cognate DNA element. Experiments aimed at determining the potential role of the 7-methylguanosine cap on RNA-binding reveal no significant difference in the affinity of YY1 for capped or uncapped mRNA. Taken together, the results show that the YY1–RNA interaction is highly stable, and that YY1 possesses the ability to interact with structurally divergent RNA substrates. These data are the first to specifically document the interaction between YY1 and potential in vivo targets.

Biochemical characterization of bona fide polycystin-1 in vitro and in vivo

2001 ◽  
Vol 38 (6) ◽  
pp. 1421-1429 ◽  
Author(s):  
Alessandra Boletta ◽  
Feng Qian ◽  
Luiz F. Onuchic ◽  
Alessandra Bragonzi ◽  
Marina Cortese ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Bona Fide

scholarly journals RNA triphosphatase and guanylyl transferase activities are associated with the RNA polymerase protein L of rinderpest virus

2009 ◽  
Vol 90 (7) ◽  
pp. 1748-1756 ◽  
Author(s):  
M. Gopinath ◽  
M. S. Shaila
Keyword(s):  
Biochemical Characterization ◽  
Covalent Bond ◽  
Rinderpest Virus ◽  
Viral Mrnas ◽  
L Protein ◽  
Rna Triphosphatase ◽  
Rnp Complex

Rinderpest virus (RPV) large (L) protein is an integral part of the ribonucleoprotein (RNP) complex of the virus that is responsible for transcription and replication of the genome. Previously, we have shown that recombinant L protein coexpressed along with P protein (as the L–P complex) catalyses the synthesis of all viral mRNAs in vitro and the abundance of mRNAs follows a gradient of polarity, similar to the occurrence in vivo. In the present work, we demonstrate that the viral mRNAs synthesized in vitro by the recombinant L or purified RNP are capped and methylated at the N7 guanine position. RNP from the purified virions, as well as recombinant L protein, shows RNA triphosphatase (RTPase) and guanylyl transferase (GT) activities. L protein present in the RNP complex catalyses the removal of γ-phosphate from triphosphate-ended 25 nt RNA generated in vitro representing the viral N-terminal mRNA 5′ sequence. The L protein forms a covalent enzyme–guanylate intermediate with the GMP moiety of GTP, whose formation is inhibited by the addition of pyrophosphate; thus, it exhibits characteristics of cellular GTs. The covalent bond between the enzyme and nucleotide is acid labile and alkali stable, indicating the presence of phosphoamide linkage. The C-terminal region (aa 1717–2183) of RPV L protein alone exhibits the first step of GT activity needed to form a covalent complex with GMP, though it lacks the ability to transfer GMP to substrate RNA. Here, we describe the biochemical characterization of the newly found RTPase/GT activity of L protein.

scholarly journals Genetic and Biochemical Characterization of the Poly(3-Hydroxybutyrate-co-3-Hydroxyvalerate) Synthase in Haloferax mediterranei

2008 ◽  
Vol 190 (12) ◽  
pp. 4173-4180 ◽  
Author(s):  
Qiuhe Lu ◽  
Jing Han ◽  
Ligang Zhou ◽  
Jian Zhou ◽  
Hua Xiang
Keyword(s):  
Biochemical Characterization ◽  
Complete Loss ◽  
Pha Synthase ◽  
Significant Activity ◽  
Class Iii ◽  
Haloferax Mediterranei ◽  
Thermal Asymmetric Interlaced Pcr

ABSTRACT The haloarchaeon Haloferax mediterranei has shown promise for the economical production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), a desirable bioplastic. However, little is known at present about the genes involved in PHBV synthesis in the domain Archaea. In this study, we cloned the gene cluster (phaEC Hme) encoding a polyhydroxyalkanoate (PHA) synthase in H. mediterranei CGMCC 1.2087 via thermal asymmetric interlaced PCR. Western blotting revealed that the phaE Hme and phaC Hme genes were constitutively expressed, and both the PhaEHme and PhaCHme proteins were strongly bound to the PHBV granules. Interestingly, CGMCC 1.2087 could synthesize PHBV in either nutrient-limited medium (supplemented with 1% starch) or nutrient-rich medium, up to 24 or 18% (wt/wt) in shaking flasks. Knockout of the phaEC Hme genes in CGMCC 1.2087 led to a complete loss of PHBV synthesis, and only complementation with the phaEC Hme genes together (but not either one alone) could restore to this mutant the capability for PHBV accumulation. The known haloarchaeal PhaC subunits are much longer at their C termini than their bacterial counterparts, and the C-terminal extension of PhaCHme was proven to be indispensable for its function in vivo. Moreover, the mixture of purified PhaEHme/PhaCHme (1:1) showed significant activity of PHA synthase in vitro. Taken together, our results indicated that a novel member of the class III PHA synthases, composed of PhaCHme and PhaEHme, accounted for the PHBV synthesis in H. mediterranei.

Biochemical Characterization of Chloromethane Emission from the Wood-Rotting Fungus Phellinus pomaceus

1998 ◽  
Vol 64 (8) ◽  
pp. 2831-2835 ◽  
Author(s):  
Deepti Saxena ◽  
Saleh Aouad ◽  
Jihad Attieh ◽  
Hargurdeep S. Saini
Keyword(s):  
Atmospheric Chemistry ◽  
Biochemical Characterization ◽  
Active Form ◽  
Methyl Chloride ◽  
Bound State ◽  
Ph Optimum ◽  
Methyl Donors ◽  
Membrane Bound

ABSTRACT Many wood-rotting fungi, including Phellinus pomaceus, produce chloromethane (CH3Cl). P. pomaceus can be cultured in undisturbed glucose mycological peptone liquid medium to produce high amounts of CH3Cl. The biosynthesis of CH3Cl is catalyzed by a methyl chloride transferase (MCT), which appears to be membrane bound. The enzyme is labile upon removal from its natural location and upon storage at low temperature in its bound state. Various detergents failed to solubilize the enzyme in active form, and hence it was characterized by using a membrane fraction. The enzyme had a sharp pH optimum between 7 and 7.2. Its apparent Km for Cl− (ca. 300 mM) was much higher than that for I− (250 μM) or Br− (11 mM). A comparison of theseKm values to the relative in vivo methylation rates for different halides suggests that the realKm for Cl− may be much lower, but the calculated value is high because the CH3Cl produced is used immediately in a coupled reaction. Among various methyl donors tested, S-adenosyl-l-methionine (SAM) was the only one that supported significant methylation by MCT. The reaction was inhibited by S-adenosyl-l-homocysteine, an inhibitor of SAM-dependent methylation, suggesting that SAM is the natural methyl donor. These findings advance our comprehension of a poorly understood metabolic sector at the origin of biogenic emissions of halomethanes, which play an important role in atmospheric chemistry.

GENETIC LOCALIZATION AND BIOCHEMICAL CHARACTERIZATION OF A TRANS-ACTING REGULATORY EFFECT IN DROSOPHILA

Genetics ◽  
1983 ◽  
Vol 105 (1) ◽  
pp. 55-69
Author(s):  
Joseph J King ◽  
John F McDonald
Keyword(s):  
Drosophila Melanogaster ◽  
Biochemical Characterization ◽  
Regulatory Gene ◽  
Regulatory Effect ◽  
Protein Levels ◽  
The Third ◽  
Genetic Localization ◽  
Glycerophosphate Dehydrogenase

ABSTRACT A region-specific, trans-acting regulatory gene that alters in vivo protein levels of α-glycerophosphate dehydrogenase (α-GPDH) has been mapped to position 55.4 on the third chromosome of Drosophila melanogaster. The gene has been found to affect the in vivo stability of α-GPDH in adult thoracic tissue but has no effect on α-GPDH levels in the abdomen. Although no other thoracic proteins were found to be influenced by the locus, it appears to modify the level of one additional abdominal protein. The action of the gene over development and its possible mode of control are discussed.

scholarly journals The Human FSGS-Causing ANLN R431C Mutation Induces Dysregulated PI3K/AKT/mTOR/Rac1 Signaling in Podocytes

2018 ◽  
Vol 29 (8) ◽  
pp. 2110-2122 ◽  
Author(s):  
Gentzon Hall ◽  
Brandon M. Lane ◽  
Kamal Khan ◽  
Igor Pediaditakis ◽  
Jianqiu Xiao ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Adaptor Protein ◽  
Pi3k Pathway ◽  
Actin Binding ◽  
Loss Of Function ◽  
Signaling Axis ◽  
Phosphoinositide 3 Kinase

BackgroundWe previously reported that mutations in the anillin (ANLN) gene cause familial forms of FSGS. ANLN is an F-actin binding protein that modulates podocyte cell motility and interacts with the phosphoinositide 3-kinase (PI3K) pathway through the slit diaphragm adaptor protein CD2-associated protein (CD2AP). However, it is unclear how the ANLN mutations cause the FSGS phenotype. We hypothesized that the R431C mutation exerts its pathogenic effects by uncoupling ANLN from CD2AP.MethodsWe conducted in vivo complementation assays in zebrafish to determine the effect of the previously identified missense ANLN variants, ANLNR431C and ANLNG618C during development. We also performed in vitro functional assays using human podocyte cell lines stably expressing wild-type ANLN (ANLNWT) or ANLNR431C.ResultsExperiments in anln-deficient zebrafish embryos showed a loss-of-function effect for each ANLN variant. In human podocyte lines, expression of ANLNR431C increased cell migration, proliferation, and apoptosis. Biochemical characterization of ANLNR431C-expressing podocytes revealed hyperactivation of the PI3K/AKT/mTOR/p70S6K/Rac1 signaling axis and activation of mTOR-driven endoplasmic reticulum stress in ANLNR431C-expressing podocytes. Inhibition of mTOR, GSK-3β, Rac1, or calcineurin ameliorated the effects of ANLNR431C. Additionally, inhibition of the calcineurin/NFAT pathway reduced the expression of endogenous ANLN and mTOR.ConclusionsThe ANLNR431C mutation causes multiple derangements in podocyte function through hyperactivation of PI3K/AKT/mTOR/p70S6K/Rac1 signaling. Our findings suggest that the benefits of calcineurin inhibition in FSGS may be due, in part, to the suppression of ANLN and mTOR. Moreover, these studies illustrate that rational therapeutic targets for familial FSGS can be identified through biochemical characterization of dysregulated podocyte phenotypes.

Saliva and Dental Pellicle-A Review

2000 ◽  
Vol 14 (1) ◽  
pp. 22-28 ◽  
Author(s):  
U. Lendenmann ◽  
J. Grogan ◽  
F.G. Oppenheim
Keyword(s):  
Biochemical Characterization ◽  
Tooth Enamel ◽  
Selective Adsorption ◽  
Salivary Proteins ◽  
Single Donor ◽  
Composition And Structure ◽  
Acquired Enamel Pellicle

The acquired enamel pellicle is an organic film covering the surfaces of teeth. When this film was first discovered, it was thought to be of embryologic origin. Only in the middle of this century did it become clear that it was acquired after tooth eruption. Initially, the small amounts of material that could be obtained have virtually limited the investigation of pellicle proteins to amino acid analysis. Nevertheless, this technique revealed that the pellicle is mainly proteinaceous and is formed by selective adsorption of salivary proteins on tooth enamel. Later, immunologic techniques allowed for the identification of many salivary and fewer non-salivary proteins as constituents of pellicle. However, to this date, isolation and direct biochemical characterization of in vivo pellicle protein have not been possible, because only a few micrograms can be obtained from a single donor. Therefore, the composition and structure of the acquired enamel pellicle are still essentially unknown. Information on the functions of pellicle has been obtained mainly from in vitro experiments carried out with saliva-coated hydroxyapatite and enamel discs. It was found that pellicle protects enamel by reducing demineralization upon acid challenge. Improved pellicle harvesting procedures and analysis by state-of-the-art proteomics with mass spectroscopy approaches promise to make major inroads into the characterization of enamel pellicle.

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