scholarly journals Role of pfkA and General Carbohydrate Catabolism in Seed Colonization by Enterobacter cloacae

1999 ◽  
Vol 65 (6) ◽  
pp. 2513-2519 ◽  
Author(s):  
D. P. Roberts ◽  
P. D. Dery ◽  
I. Yucel ◽  
J. Buyer ◽  
M. A. Holtman ◽  
...  
Keyword(s):  
Amino Acids ◽  
Sweet Corn ◽  
Carbon Sources ◽  
Enterobacter Cloacae ◽  
In Vitro Growth ◽  
Wild Type ◽  
Exogenous Addition ◽  
Total Carbohydrates ◽  
Pea Seeds

ABSTRACT Enterobacter cloacae A-11 is a transposon mutant of strain 501R3 that was deficient in cucumber spermosphere colonization and in the utilization of certain carbohydrates (D. P. Roberts, C. J. Sheets, and J. S. Hartung, Can. J. Microbiol. 38:1128–1134, 1992). In vitro growth of strain A-11 was reduced or deficient on most carbohydrates that supported growth of strain 501R3 but was unaffected on fructose, glycerol, and all amino acids and organic acids tested. Colonization by strain A-11 was significantly reduced (P ≤ 0.05) for cucumber and radish seeds compared to that of strain 501R3, but colonization of pea, soybean, sunflower, and sweet corn seeds was not reduced. Pea seeds released several orders of magnitude more total carbohydrates and amino acids than cucumber and radish seeds and approximately 4,000-fold more fructose. Fructose was the only carbohydrate detected in the seed exudates which supported wild-type levels of in vitro growth of strain A-11. Soybean, sunflower, and sweet corn seeds also released significantly greater amounts of fructose and total carbohydrates and amino acids than cucumber or radish seeds. The exogenous addition of fructose to cucumber and radish seeds at quantities similar to the total quantity of carbohydrates released from pea seeds over 96 h increased the populations of strain A-11 to levels comparable to those of strain 501R3 in sterile sand. Molecular characterization of strain A-11 indicated that the mini-Tn5 kanamycin transposon was inserted in a region of the genome with significant homology topfkA, which encodes phosphofructo kinase. A comparison of strain A-11 with Escherichia coli DF456, a knownpfkA mutant, indicated that the nutritional loss phenotypes were identical. Furthermore, the pfkA homolog cloned fromE. cloacae 501R3 complemented the nutritional loss phenotypes of both E. coli DF456 and E. cloacaeA-11 and restored colonization by strain A-11 to near wild-type levels. These genetic and biochemical restoration experiments provide strong evidence that the quantities of reduced carbon sources found in seed exudates and the ability of microbes to use these compounds play important roles in the colonization of the spermosphere.

THE IN VITRO RELEASE OF AMINO ACIDS BY RUMEN PAPILLAE

1980 ◽  
Vol 60 (2) ◽  
pp. 281-291 ◽  
Author(s):  
R. J. BOILA ◽  
L. P. MILLIGAN
Keyword(s):  
Amino Acids ◽  
Carbon Sources ◽  
In Vitro Release ◽  
Nitrogen Sources ◽  
Chromatographic Technique ◽  
Salts Of Organic Acids ◽  
Liquid Chromatographic ◽  
Vitro Release ◽  
Effective Source

Rumen papillae from cattle were incubated aerobically with combinations of NH4Cl, amino acids and salts of organic acids, the latter including propionate, pyruvate, α-ketoglutarate and glyoxylate. Amino acids in the incubation media were analyzed using a gas-liquid chromatographic technique entailing separation of the isobutyl-N(0)-heptafluorobutyryl esters: glutamine was recovered with glutamate, asparagine with aspartate, and citrulline with ornithine. Rumen papillae incubated with pyruvate or propionate released alanine, but with the latter substrate only glutamate was effective as a nitrogen source. Glycine and glutamate plus glutamine were released in the presence of glyoxylate and α-ketoglutarate, respectively. Serine and aspartate plus asparagine were not quantitatively major products released by rumen papillae. Glutamate was an effective source of nitrogen for the release of alanine and glycine with pyruvate and glyoxylate, respectively, as carbon sources. When rumen papillae were incubated with pyruvate or glyoxylate as the added carbon source, glutamine nitrogen disappeared and was not accounted for by the amino acids measured. With arginine as a substrate, there was a release of ornithine by rumen papillae indicating urea production. The tissues of rumen papillae appear to synthesize amino acids from expected carbon sources with ammonia or glutamate as nitrogen sources and to catabolize glutamine and arginine. The metabolism of amino acids by rumen papillae would contribute to the interchange of nitrogen between the rumen and the host.

scholarly journals The anti-inflammatory peptide Ac-SDKP is released from thymosin-β4 by renal meprin-α and prolyl oligopeptidase

2016 ◽  
Vol 310 (10) ◽  
pp. F1026-F1034 ◽  
Author(s):  
Nitin Kumar ◽  
Pablo Nakagawa ◽  
Branislava Janic ◽  
Cesar A. Romero ◽  
Morel E. Worou ◽  
...  
Keyword(s):  
Amino Acids ◽  
Rat Kidney ◽  
Plasma Concentrations ◽  
Potential Candidate ◽  
Thymosin Β4 ◽  
Wild Type ◽  
Prolyl Oligopeptidase ◽  
Anti Inflammatory

N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) is a natural tetrapeptide with anti-inflammatory and antifibrotic properties. Previously, we have shown that prolyl oligopeptidase (POP) is involved in the Ac-SDKP release from thymosin-β4 (Tβ4). However, POP can only hydrolyze peptides shorter than 30 amino acids, and Tβ4 is 43 amino acids long. This indicates that before POP hydrolysis takes place, Tβ4 is hydrolyzed by another peptidase that releases NH2-terminal intermediate peptide(s) with fewer than 30 amino acids. Our peptidase database search pointed out meprin-α metalloprotease as a potential candidate. Therefore, we hypothesized that, prior to POP hydrolysis, Tβ4 is hydrolyzed by meprin-α. In vitro, we found that the incubation of Tβ4 with both meprin-α and POP released Ac-SDKP, whereas no Ac-SDKP was released when Tβ4 was incubated with either meprin-α or POP alone. Incubation of Tβ4 with rat kidney homogenates significantly released Ac-SDKP, which was blocked by the meprin-α inhibitor actinonin. In addition, kidneys from meprin-α knockout (KO) mice showed significantly lower basal Ac-SDKP amount, compared with wild-type mice. Kidney homogenates from meprin-α KO mice failed to release Ac-SDKP from Tβ4. In vivo, we observed that rats treated with the ACE inhibitor captopril increased plasma concentrations of Ac-SDKP, which was inhibited by the coadministration of actinonin (vehicle, 3.1 ± 0.2 nmol/l; captopril, 15.1 ± 0.7 nmol/l; captopril + actinonin, 6.1 ± 0.3 nmol/l; P < 0.005). Similar results were obtained with urinary Ac-SDKP after actinonin treatment. We conclude that release of Ac-SDKP from Tβ4 is mediated by successive hydrolysis involving meprin-α and POP.

scholarly journals Effects of Various Carbon Sources and Their Combinations on in vitro Growth and Photosynthesis of Banana Plantlets

2000 ◽  
Vol 3 (4) ◽  
pp. 392-397 ◽  
Author(s):  
John Nelson Buah ◽  
Yoshinobu Kawamitsu ◽  
Shigeyasu Yonemori ◽  
Mitsuru Hayashi ◽  
Seiichi Murayama
Keyword(s):  
Carbon Sources ◽  
In Vitro Growth

Amino acids as reduced carbon sources for Enterobacter cloacae during colonization of the spermospheres of crop plants

1996 ◽  
Vol 28 (8) ◽  
pp. 1015-1020 ◽  
Author(s):  
D.P. Roberts ◽  
A.M. Marty ◽  
P.D. Dery ◽  
I. Yucel ◽  
J.S. Hartung
Keyword(s):  
Amino Acids ◽  
Carbon Sources ◽  
Enterobacter Cloacae ◽  
Crop Plants

scholarly journals A C-terminal Hydrophobic Region is Required for Homo-Oligomerization of the Hepatitis E Virus Capsid (ORF2) Protein

2001 ◽  
Vol 1 (3) ◽  
pp. 122-128 ◽  
Author(s):  
Li Xiaofang ◽  
Mohammad Zafrullah ◽  
Faizan Ahmad ◽  
Shahid Jameel
Keyword(s):  
Amino Acids ◽  
Hepatitis E Virus ◽  
Hepatitis E ◽  
Wild Type ◽  
Hydrophobic Region ◽  
Reading Frame ◽  
Orf2 Protein ◽  
Acute Form ◽  
Open Reading Frame 2

Hepatitis E virus (HEV) is the causative agent of hepatitis E, an acute form of viral hepatitis. The open reading frame 2 (ORF2) of HEV encodes the viral capsid protein, which can self-oligomerize into virus-like particles. To understand the domains within this protein important for capsid biogenesis, we have carried out in vitro analyses of association and folding patterns of wild type and mutant ORF2 proteins. When expressedin vitroor in transfected cells, the ORF2 protein assembled as dimers, trimers and higher order forms. While N-terminal deletions upto 111 amino acids had no effect, the deletion of amino acids 585–610 led to reduced homo-oligomerization. This deletion also resulted in aberrant folding of the protein, as determined by its sensitivity to trypsin. This study suggests that a C-terminal hydrophobic region encompassing amino acids 585–610 of the ORF2 protein might be critical for capsid biogenesis.

scholarly journals The Slow Wallerian Degeneration Protein, WldS, Binds Directly to VCP/p97 and Partially Redistributes It within the Nucleus

2006 ◽  
Vol 17 (3) ◽  
pp. 1075-1084 ◽  
Author(s):  
Heike Laser ◽  
Laura Conforti ◽  
Giacomo Morreale ◽  
Till G.M. Mack ◽  
Molly Heyer ◽  
...  
Keyword(s):  
Amino Acids ◽  
Wallerian Degeneration ◽  
Nuclear Protein ◽  
Ubiquitin Proteasome System ◽  
Wild Type ◽  
Ubiquitin Proteasome ◽  
Direct Binding ◽  
Synthesis Activity ◽  
Additional Role

Slow Wallerian degeneration (WldS) mutant mice express a chimeric nuclear protein that protects sick or injured axons from degeneration. The C-terminal region, derived from NAD+ synthesizing enzyme Nmnat1, is reported to confer neuroprotection in vitro. However, an additional role for the N-terminal 70 amino acids (N70), derived from multiubiquitination factor Ube4b, has not been excluded. In wild-type Ube4b, N70 is part of a sequence essential for ubiquitination activity but its role is not understood. We report direct binding of N70 to valosin-containing protein (VCP; p97/Cdc48), a protein with diverse cellular roles including a pivotal role in the ubiquitin proteasome system. Interaction with WldS targets VCP to discrete intranuclear foci where ubiquitin epitopes can also accumulate. WldS lacking its N-terminal 16 amino acids (N16) neither binds nor redistributes VCP, but continues to accumulate in intranuclear foci, targeting its intrinsic NAD+ synthesis activity to these same foci. Wild-type Ube4b also requires N16 to bind VCP, despite a more C-terminal binding site in invertebrate orthologues. We conclude that N-terminal sequences of WldS protein influence the intranuclear location of both ubiquitin proteasome and NAD+ synthesis machinery and that an evolutionary recent sequence mediates binding of mammalian Ube4b to VCP.

scholarly journals Mutations within the Putative Membrane-Spanning Domain of the Simian Immunodeficiency Virus Transmembrane Glycoprotein Define the Minimal Requirements for Fusion, Incorporation, and Infectivity

2001 ◽  
Vol 75 (20) ◽  
pp. 9601-9612 ◽  
Author(s):  
John T. West ◽  
Patrick B. Johnston ◽  
Susan R. Dubay ◽  
Eric Hunter
Keyword(s):  
Amino Acids ◽  
Cell Fusion ◽  
Virus Replication ◽  
Expression Vectors ◽  
Wild Type ◽  
Short Delay ◽  
Minimum Requirement ◽  
Helical Turn ◽  
Membrane Spanning

ABSTRACT The membrane-spanning domain (MSD) of a number of retroviral transmembrane (TM) glycoproteins, including those from the human and simian immunodeficiency viruses (HIV and SIV), have been predicted to contain a charged arginine residue. The wild-type SIV TM glycoprotein is 354 amino acids long. The entire putative cytoplasmic domain of SIV (amino acids 193 to 354) is dispensable for virus replication in vitro, and such truncation-containing viruses are capable of reaching wild-type titers after a short delay. We show here that further truncation of eight additional amino acids to TM185 results in a protein that lacks fusogenicity but is, nevertheless, efficiently incorporated into budding virions. By analyzing a series of nonsense mutations between amino acids 193 and 185 in Env expression vectors and in the SIVmac239 proviral clone, a region of the SIV TM that contains the minimum requirement for glycoprotein-mediated cell-to-cell fusion and that for virus replication was identified. Virus entry and infectivity were evident in truncations to a minimum of 189 amino acids, whereas cell-cell fusion was observed for a protein of only 187 amino acids. Glycoprotein was efficiently incorporated into budding virions in truncations up to 185 amino acids, indicating that such proteins are membrane anchored and are transported to the cell surface. However, truncation of the TM to 180 amino acids resulted in a protein that displays a transport defect and may be retained in the endoplasmic reticulum. Based on our analyses of these mutants, an alternative model for the MSD of SIV is proposed. Our model suggests that membrane-imbedded charged residues can be neutralized by side-chain interactions with lipid polar head groups. As a consequence, the membrane-spanning region can be reduced by more than a helical turn. This new model accounts for the ability of truncations within the predicted MSD to remain membrane anchored and maintain biological activity.

scholarly journals Cell surface expression of v-fms-coded glycoproteins is required for transformation.

1984 ◽  
Vol 4 (10) ◽  
pp. 1999-2009 ◽  
Author(s):  
M F Roussel ◽  
C W Rettenmier ◽  
A T Look ◽  
C J Sherr
Keyword(s):  
Amino Acids ◽  
Protein Kinase ◽  
Cell Surface ◽  
Gene Product ◽  
Kinase Activity ◽  
Specific Protein ◽  
Protein Kinase Activity ◽  
Wild Type ◽  
Specific Protein Kinase

The viral oncogene v-fms encodes a transforming glycoprotein with in vitro tyrosine-specific protein kinase activity. Although most v-fms-coded molecules remain internally sequestered in transformed cells, a minor population of molecules is transported to the cell surface. An engineered deletion mutant lacking 348 base pairs of the 3.0-kilobase-pair v-fms gene encoded a polypeptide that was 15 kilodaltons smaller than the wild-type v-fms gene product. The in-frame deletion of 116 amino acids was adjacent to the transmembrane anchor peptide located near the middle of the predicted protein sequence and 432 amino acids from the carboxyl terminus. The mutant polypeptide acquired N-linked oligosaccharide chains, was proteolytically processed in a manner similar to the wild-type glycoprotein, and exhibited an associated tyrosine-specific protein kinase activity in vitro. However, the N-linked oligosaccharides of the mutant glycoprotein were not processed to complex carbohydrate chains, and the glycoprotein was not detected at the cell surface. Cells expressing high levels of the mutant glycoprotein did not undergo morphological transformation and did not form colonies in semisolid medium. The transforming activity of the v-fms gene product therefore appears to be mediated through target molecules on the plasma membrane.

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