Monomeric and dimeric GDF-5 show equal type I receptor binding and oligomerization capability and have the same biological activity

2006 ◽  
Vol 387 (4) ◽  
pp. 451-460 ◽  
Author(s):  
Christina Sieber ◽  
Frank Plöger ◽  
Raphaela Schwappacher ◽  
Rolf Bechtold ◽  
Michael Hanke ◽  
...  
Keyword(s):  
Biological Activity ◽  
Biochemical Analysis ◽  
Gene Activation ◽  
Disulfide Bridge ◽  
Living Cells ◽  
Type I ◽  
E Coli ◽  
Interchain Disulfide Bond ◽  
Dimeric Form

Abstract Growth and differentiation factor 5 (GDF-5) is a homodimeric protein stabilized by a single disulfide bridge between cysteine 465 in the respective monomers, as well as by three intramolecular cysteine bridges within each subunit. A mature recombinant human GDF-5 variant with cysteine 465 replaced by alanine (rhGDF-5 C465A) was expressed in E. coli, purified to homogeneity, and chemically renatured. Biochemical analysis showed that this procedure eliminated the sole interchain disulfide bond. Surprisingly, the monomeric variant of rhGDF-5 is as potent in vitro as the dimeric form. This could be confirmed by alkaline phosphatase assays and Smad reporter gene activation. Furthermore, dimeric and monomeric rhGDF-5 show comparable binding to their specific type I receptor, BRIb. Studies on living cells showed that both the dimeric and monomeric rhGDF-5 induce homomeric BRIb and heteromeric BRIb/BRII oligomers. Our results suggest that rhGDF-5 C465A has the same biological activity as rhGDF-5 with respect to binding to, oligomerization of and signaling through the BMP receptor type Ib.

scholarly journals PHAGOCYTOSIS OF BACTERIA IN THE ABSENCE OF ANTIBODY AND THE EFFECT OF PHYSICAL SURFACE

1956 ◽  
Vol 104 (2) ◽  
pp. 233-243 ◽  
Author(s):  
Edwin M. Lerner
Keyword(s):  
Filter Paper ◽  
Chemical Effect ◽  
Type I ◽  
Blood Leukocytes ◽  
Experimental Conditions ◽  
Phagocytic Capacity ◽  
E Coli ◽  
Physical Surface ◽  
Type Iv

The present experiments have shown that phagocytosis occurs in the absence of specific antibody and in the absence of a "suitable physical surface", as further that the presence of a rough surface does not increase the in vitro phagocytosis of pneumococci by polymorphonuclear leukocytes. This held true during repetition of Wood's experiments, as well as when more controlled quantitative techniques were employed, when conditions were made optimal for phagocytosis by increasing bacterial concentrations, and when blood leukocytes were substituted for exudate leukocytes. Evidence has been presented previously that the stimulation of phagocytosis of E. coli, B. abortus, and Type IV Pneumococcus, after contact with filter paper or an active compound present in filter paper, is a chemical effect rather than a physical effect. This type of stimulation did not occur with the Type I A5 Pneumococcus. The leukocyte of the circulating blood was found to be definitely superior to the exudate leukocyte in phagocytic capacity, under all the experimental conditions tested.

Synthesis and in vitro Biological Activity of New 4,6-Disubstituted 3(2H)-Pyridazinone-acetohydrazide Derivatives

2012 ◽  
Vol 67 (5-6) ◽  
pp. 257-265
Author(s):  
Murat Sukuroglu ◽  
Tijen Onkol ◽  
Fatma Kaynak Onurdağ ◽  
Gulsen Akalın ◽  
M. Fethi Şahin
Keyword(s):  
Biological Activity ◽  
Antimicrobial Activities ◽  
Cytotoxic Activities ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
E Coli ◽  
Preliminary Results ◽  
H Nmr ◽  
Pyridazinone Derivatives

New 3(2H)-pyridazinone derivatives containing a N’-benzyliden-acetohydrazide moiety at position 2 were synthesized. The structures of these newly synthesized compounds were confi rmed by IR, 1H NMR, and MS data. These compounds were tested for their antibacterial, antifungal, antimycobacterial, and cytotoxic activities. The compounds 2-[4-(4-chlorophenyl)- 6-(morpholin-4-yl)-3-oxo-(2H)-pyridazin-2-yl]-N’-(4-tert-butylbenzyliden)acetohydrazide and 2-[4-(4-chlorophenyl)-6-(morpholin-4-yl)-3-oxo-(2H)-pyridazin-2-yl]-N’-(4-chlorobenzyliden) acetohydrazide exhibited activity against both Gram-positive and Gram-negative bacteria. Most of the compounds were active against E. coli ATCC 35218. The preliminary results of this study revealed that some target compounds exhibited promising antimicrobial activities

scholarly journals Antimicrobial effects of fruit sauces on some pathogenic bacteria in vitro and on chicken breast meat

2021 ◽  
Vol 72 (1) ◽  
pp. 2703
Author(s):  
I VAR ◽  
S UZUNLU ◽  
I DEĞIRMENCI
Keyword(s):  
Antimicrobial Activity ◽  
Pathogenic Bacteria ◽  
Diffusion Method ◽  
Chicken Breast ◽  
Type I ◽  
Antimicrobial Effects ◽  
E Coli ◽  
Agar Diffusion

The use of natural food additives is currently a rising trend. In the present study, the aim was to determine the antimicrobial effects of plum, pomegranate, Seville orange and sumac sauces on E. coli O157:H7,E. coli type I,Listeriamonocytogenes, Listeria ivanovii, Salmonella Typhimurium and Staphylococcus aureus. Different concentrations (1%, 10%, 100%, v/v) of the sauces were tested on the studied bacteria in vitro using the agar diffusion and minimal inhibition concentration (MIC) methods. The results showed that the sumac sauce had the highest antimicrobial activity. The Seville orange, plum and pomegranate sauces also exerted antimicrobial activity in descending order. The antimicrobial activity of the fruit sauces was more effective at a concentration of 100% than at 10% and 1%, v/v. The most inhibitory effect was recorded for sumac sauce at a concentration of 100% (v/v) on L.monocytogenesand E. coli O157:H7. The findings of the MIC method aligned with the agar diffusion method. In addition, the in situ(food method) antimicrobial effect of the sauces on the indigenous microflora of chicken breast samples sold in stores was determined. Chicken samples hosting aerobic mesophilic bacteria, coliforms and E. coli were treated for two hours at 4 °C with plum, pomegranate, Seville orange and sumac sauces and were then monitored. The findings revealed that the Seville orange and sumac sauces were the most effective in reducing the indigenous microbial growth on the chicken samples. The plum sauce showed higher antimicrobial activity than pomegranate sauce. The phenolic content and acidity of the samples significantly (P< 0.05) affected the antimicrobial activity both in vitro (agar diffusion and MIC) and in situ (chilled chicken breast). In conclusion, the sumac and Seville orange sauces were found to be the most promising natural antibacterial agents, and their use could be recommended, for example, in catering services to reduce the risk of foodborne illness.

scholarly journals Cardiolipin is an Optimal Phospholipid for the Assembly, Stability, and Proper Functionality of the Dimeric Form of NhaA Na+/H+ Antiporter

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Abraham Rimon ◽  
Ramakanta Mondal ◽  
Assaf Friedler ◽  
Etana Padan
Keyword(s):  
Binding Site ◽  
The Other ◽  
Phosphatidyl Glycerol ◽  
Dimer Interface ◽  
Future Studies ◽  
E Coli ◽  
Lipid Interactions ◽  
Dimeric Form

AbstractCardiolipin (CL) was shown to bound to the dimer interface of NhaA Na+/H+ antiporter. Here, we explore the cardiolipin-NhaA interaction both in vitro and in vivo. Using a novel and straightforward in-vitro assay in which n-dodecyl β-D maltoside (DDM) detergent is used to delipidate the dimer interface and to split the dimers into monomers; the monomers are subsequently exposed to cardiolipin or the other E. coli phospholipids. Most efficient reconstitution of dimers is observed by cardiolipin. This assay is likely to be applicable to future studies of protein–lipid interactions. In-vivo experiments further reveal that cardiolipin is necessary for NhaA survival. Although less efficient phosphatidyl-glycerol (PG) can also reconstitute NhaA monomers to dimers. We also identify a putative cardiolipin binding site. Our observations may contribute to drug design, as human NhaA homologues, which are involved in severe pathologies, might also require specific phospholipids.

Myeloid cell-specific inositol polyphosphate-4-phosphatase type I knockout mice impair bacteria clearance in a murine peritonitis model

2016 ◽  
Vol 22 (6) ◽  
pp. 444-451 ◽  
Author(s):  
Shin Morioka ◽  
Kiyomi Nigorikawa ◽  
Junko Sasaki ◽  
Kaoru Hazeki ◽  
Yoshihiro Kasuu ◽  
...  
Keyword(s):  
Inflammatory Cytokines ◽  
Knockout Mice ◽  
Bacterial Infections ◽  
Myeloid Cell ◽  
Conditional Knockout ◽  
Type I ◽  
Inositol Polyphosphate ◽  
Akt Phosphorylation ◽  
E Coli

Phosphatidylinositol 3-kinase (PI3K)/Akt signaling has been implicated in the anti-inflammatory response in a mouse model of endotoxemia and sepsis. The present study focused on the role of inositol polyphosphate-4-phosphatase type I (Inpp4a), which dephosphorylates PtdIns(3,4)P2 to PtdIns(3)P, in bacterial infections. We prepared myeloid cell-specific Inpp4a-conditional knockout mice. Macrophages from these mice showed increased Akt phosphorylation and reduced production of inflammatory cytokines in response to LPS or Escherichia coli in vitro. The Inpp4a knockout mice survived for a shorter time than wild type mice after i.p. infection with E. coli, with less production of inflammatory cytokines. Additionally, E. coli clearance from blood and lung was significantly impaired in the knockout mice. A likely mechanism is that the Inpp4a-catalyzed dephosphorylation of PtdIns(3,4)P2 down-regulates Akt pathways, which, in turn, increases the production of inflammatory mediators. This mechanism at least fits the decreased E. coli clearance and short survival in the Inpp4a knockout mice.

scholarly journals In vitro reconstruction and analysis of evolutionary variation of the tomato acylsucrose metabolic network

2015 ◽  
Vol 113 (2) ◽  
pp. E239-E248 ◽  
Author(s):  
Pengxiang Fan ◽  
Abigail M. Miller ◽  
Anthony L. Schilmiller ◽  
Xiaoxiao Liu ◽  
Itai Ofner ◽  
...  
Keyword(s):  
Biosynthetic Pathway ◽  
Biochemical Analysis ◽  
In Vitro Mutagenesis ◽  
Type I ◽  
Wild Tomato Species ◽  
Tomato Species ◽  
Insect Defense ◽  
Evolutionary Variation ◽  
Ester Substrate

Plant glandular secreting trichomes are epidermal protuberances that produce structurally diverse specialized metabolites, including medically important compounds. Trichomes of many plants in the nightshade family (Solanaceae) produce O-acylsugars, and in cultivated and wild tomatoes these are mixtures of aliphatic esters of sucrose and glucose of varying structures and quantities documented to contribute to insect defense. We characterized the first two enzymes of acylsucrose biosynthesis in the cultivated tomato Solanum lycopersicum. These are type I/IV trichome-expressed BAHD acyltransferases encoded by Solyc12g006330─or S. lycopersicum acylsucrose acyltransferase 1 (Sl-ASAT1)─and Solyc04g012020 (Sl-ASAT2). These enzymes were used—in concert with two previously identified BAHD acyltransferases—to reconstruct the entire cultivated tomato acylsucrose biosynthetic pathway in vitro using sucrose and acyl-CoA substrates. Comparative genomics and biochemical analysis of ASAT enzymes were combined with in vitro mutagenesis to identify amino acids that influence CoA ester substrate specificity and contribute to differences in types of acylsucroses that accumulate in cultivated and wild tomato species. This work demonstrates the feasibility of the metabolic engineering of these insecticidal metabolites in plants and microbes.

Subtype switching of ANP receptors during in vitro culture of vascular cells

1989 ◽  
Vol 256 (1) ◽  
pp. H311-H314
Author(s):  
K. Uchida ◽  
T. Mizuno ◽  
M. Shimonaka ◽  
N. Sugiura ◽  
H. Hagiwara ◽  
...  
Keyword(s):  
Biochemical Analysis ◽  
Vascular System ◽  
Jugular Vein ◽  
Type I ◽  
Type Ii ◽  
Bovine Jugular Vein ◽  
The One ◽  
Anp Receptors ◽  
Lung Receptor

Vascular receptors for atrial natriuretic peptide (ANP) have been characterized immunochemically and found to be classified into two subtypes that show differential localization and expression. An antiserum, recognizing the protein core of the receptor, was raised against the purified bovine lung ANP receptor and used for the immunochemical subtyping. ANP receptors solubilized from the bovine jugular vein reacted strongly with the antiserum as well as the homologous lung receptor; however, the receptors from the aorta and carotid artery were recognized only weakly, indicating the presence of two types of ANP receptor in the vascular system. The one that reacts strongly with the antiserum and is distributed mainly in the venous side is termed type I and the other reacting weakly and predominating in the arteries, type II. The two subtypes were also distinguishable in their ligand specificities; the type I receptor showed a remarkably higher affinity for the ANP analogue atriopeptin I than the type II receptor. Surprisingly, similar immunochemical and biochemical analysis of the receptors on cultured vascular smooth muscle and endothelial cells revealed that the arterial cells originally expressing the type II receptor begin to express the type I receptor when cultured in vitro.

scholarly journals Synthesis and characterization some complexes of azo dye of pyrimidyniyl and evaluating their biological activity

2019 ◽  
Vol 24 (3) ◽  
Author(s):  
Yareeb Jwad Sahar ◽  
Hasan Shamran Mohammed
Keyword(s):  
Biological Activity ◽  
Cell Line ◽  
Azo Dye ◽  
Mass Spectra ◽  
Molar Conductivity ◽  
Synthesis And Characterization ◽  
E Coli ◽  
Mcf 7

The present study involves fabrication a heterocyclic azo dye:  2-[4-diamino-3-benzaldehyde diazenyl]pyrimidine (L). The dye was synthesized by reacting 2-aminopyrimidine with 4-diaminobenzaldehyde (in situ). The complexes of ([Ag(L)(H2O)]NO3, [Cd(L)2]Cl2 and [Hg(L)2]Cl2) dyes were prepared and characterized by elemental analysis; mass spectra, molar conductivity and spectroscopies of infrared and UV-Vis. The L dye ligand is tridentate. The Cd(II) and Hg(II) are octahedral with mole ratio Metal:Ligand equal to 1:2 while Ag(I) complex is tetrahedral with mole ratio Metal:Ligand equal to 1:1. The molar conductivity indicates that Ag(I) complex is electrolyte (1:1) and Cd(II) and (II) are electrolytes with ratio 2:1. The synthesized ligand (L) and its complexes showed interest biological activity against E-coli and Staph-aureus bacteria and Aspergillus Niger and Pinicillium.sp fungi. The anti-activity of [Ag(L)(H2O)]NO3 complex against MCF-7 cell line and its cytotoxicity against WRL cell line were interesting in vitro.

scholarly journals Bioinformatic, Genetic, and Biochemical Evidence that Some Glycoside Hydrolase Family 42 β-Galactosidases Are Arabinogalactan Type I Oligomer Hydrolases

2006 ◽  
Vol 72 (12) ◽  
pp. 7730-7738 ◽  
Author(s):  
Stephanie Shipkowski ◽  
Jean E. Brenchley
Keyword(s):  
Glycoside Hydrolase ◽  
Glycoside Hydrolases ◽  
Glycoside Hydrolase Family ◽  
Type I ◽  
Plant Polysaccharide ◽  
E Coli ◽  
Genes Encoding ◽  
Layer Chromatography

ABSTRACT Glycoside hydrolases are organized into glycoside hydrolase families (GHFs) and within this larger group, the β-galactosidases are members of four families: 1, 2, 35, and 42. Most genes encoding GHF 42 enzymes are from prokaryotes unlikely to encounter lactose, suggesting a different substrate for these enzymes. In search of this substrate, we analyzed genes neighboring GHF 42 genes in databases and detected an arrangement implying that these enzymes might hydrolyze oligosaccharides released by GHF 53 enzymes from arabinogalactan type I, a pectic plant polysaccharide. Because Bacillus subtilis has adjacent GHF 42 and GHF 53 genes, we used it to test the hypothesis that a GHF 42 enzyme (LacA) could act on the oligosaccharides released by a GHF 53 enzyme (GalA) from galactan. We cloned these genes, plus a second GHF 42 gene from B. subtilis, yesZ, into Escherichia coli and demonstrated that cells expressing LacA with GalA gained the ability to use galactan as a carbon source. We constructed B. subtilis mutants and showed that the increased β-galactosidase activity generated in response to the addition of galactan was eliminated by inactivating lacA or galA but unaffected by the inactivation of yesZ. As further demonstration, we overexpressed the LacA and GalA proteins in E. coli and demonstrated that these enzymes degrade galactan in vitro as assayed by thin-layer chromatography. Our work provides the first in vivo evidence for a function of some GHF 42 β-galactosidases. Similar functions for other β-galactosidases in both GHFs 2 and 42 are suggested by genomic data.

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