scholarly journals Amylase and Protease Secretion by the Marine Bacterium Vibrio gazogenes

1982 ◽  
Vol 35 (4) ◽  
pp. 457 ◽  
Author(s):  
Catherine Ratcliffe ◽  
RL Sanders ◽  
C Ludmila Tittel ◽  
RW O’Brien
Keyword(s):  
Protease Activity ◽  
Metal Ion ◽  
Rna Synthesis ◽  
De Novo ◽  
Defined Medium ◽  
Logarithmic Phase ◽  
Chloromethyl Ketone ◽  
Late Logarithmic Phase ◽  
Casamino Acids ◽  
Protease Secretion

V. gazogenes secreted an amylase throughout the logarithmic phase of growth when starch or maltose was the carbon source for growth. The enzyme was apparently not constitutive and was repressed by glucose. The amylase was of the oc-type and had an optimum pH of 6·5. Protease secretion by V. gazogenes occurred when the organism was grown in a defined medium containing 0·005 % (w/v) yeast extract. The activity of the enzyme was increased 40-fold when the organism was grown in a medium containing peptone or Casamino Acids. Enzyme secretion started in the late logarithmic phase of growth and ceased when the cells entered the stationary phase of growth and was not repressed by glucose. The protease was neither induced nor repressed by glutamic acid, aspartic acid, alanine, serine, arginine, valine, threonine, lysine, leucine, proline or oc-ketoglutarate. Tryptophan delayed, but did not inhibit, protease secretion. Both the amylase and the protease were truly extracellular and were not released as a result of cell lysis. Phenylmethylsulfonyl fluoride and N-oc-p-tosyl-L-Iysine chloromethyl ketone hydrochloride, L-l-tosylamide-2-phenylethyl chloromethyl ketone, leupeptin, anti pain and chymostatin did not markedly inhibit the protease, indicating that it is not a serine protease nor similar to trypsin, chymotrypsin, papain or cathepsin B. Inhibitors of sulfhydryl enzymes were also without effect on protease activity, but 1,1O-phenanthroline, ethylenediaminetetraacetate and ethyleneglycol-bis-(p-aminoethyl ether) N,N'-tetraacetate almost completely inhibited the protease, indicating that it requires a divalent metal ion for activity. After dialysis against water, both amylase and protease lost over 90% of their activity. The amylase was almost completely reactivated by 15 mM Cl-, Be or 1-, but not by Ca 2+ or Mg2+. Protease was fully reactivated by about 30 mM Ca2+ or Mg2+ (at 70 ruM concentration these ions stimulated activity to about 140% of the rate of the undialysed enzyme). Mn2+ and Co2+ partially restored protease activity. Chloramphenicol, at concentrations that did not affect RNA synthesis, completely inhibited amylase and protease secretion, showing that secretion of both enzymes was a de novo process. When rifampin or actinomycin D, at concentrations that completely and rapidly inhibited cellular RNA synthesis, was added to cultures actively secreting amylase or protease, there was no inhibition of secretion for periods of 6-20 min, indicating the presence of a pool of mRNA specific for each enzyme. The protease was not affected by trypsin or oc-chymotrypsin during the secretion process, indicating that the enzyme had taken up its tertiary conformation before emergence from the cell membrane.

Regulation of extracellular proteases in Achlya ambisexualis

1997 ◽  
Vol 75 (3) ◽  
pp. 440-444 ◽  
Author(s):  
Terry W. Hill ◽  
Markus P. Pott
Keyword(s):  
Glutamic Acid ◽  
Molecular Mass ◽  
Protease Activity ◽  
Defined Medium ◽  
Extracellular Proteases ◽  
Nutrient Source ◽  
Achlya Ambisexualis ◽  
Extracellular Protease Activity ◽  
Protease Secretion ◽  
Secreted Enzymes

The use of proteins as a nutrient source by Achlya ambisexualis was investigated, using media containing defined and undefined sources of carbon, nitrogen, and (or) sulfur. Release of extracellular proteases occurred during growth on all proteins and protein hydrolysates tested, but not during growth on yeast extract or in defined medium. In gelatin-containing media, three major bands of extracellular protease activity were detected by electrophoresis, with estimated molecular mass of 26, 48, and 58 kDa. Growth on gelatin was stimulated to a much greater degree by the addition of glucose to the medium than by additions of glutamic acid or methionine. This and the release of ammonia during growth indicate that gelatin is less effective in meeting metabolic needs for carbon than it is in meeting the needs for nitrogen and sulfur. Protease secretion is only partially regulated by glucose, whereas glucose, methionine, and glutamic acid in combination cause almost complete repression. The pattern of regulation indicated by these results is most consistent with one of induction + derepression. Key words: oomycetes, proteinases, regulation, secreted enzymes.

Mechanism for De Novo RNA Synthesis and Initiating Nucleotide Specificity by T7 RNA Polymerase

2007 ◽  
Vol 370 (2) ◽  
pp. 256-268 ◽  
Author(s):  
William P. Kennedy ◽  
Jamila R. Momand ◽  
Y. Whitney Yin
Keyword(s):  
Rna Polymerase ◽  
Rna Synthesis ◽  
De Novo ◽  
T7 Rna Polymerase ◽  
Nucleotide Specificity

scholarly journals De Novo Genome Assembly of Limpet Bathyacmaea lactea (Gastropoda: Pectinodontidae): The First Reference Genome of a Deep-Sea Gastropod Endemic to Cold Seeps

2020 ◽  
Vol 12 (6) ◽  
pp. 905-910 ◽  
Author(s):  
Ruoyu Liu ◽  
Kun Wang ◽  
Jun Liu ◽  
Wenjie Xu ◽  
Yang Zhou ◽  
...  
Keyword(s):  
Deep Sea ◽  
Metal Ion ◽  
De Novo ◽  
Demographic History ◽  
Gene Families ◽  
Phylogenetic Position ◽  
Cold Seeps ◽  
Nitrogen And Phosphorus ◽  
De Novo Genome Assembly ◽  
A Genome

Abstract Cold seeps, characterized by the methane, hydrogen sulfide, and other hydrocarbon chemicals, foster one of the most widespread chemosynthetic ecosystems in deep sea that are densely populated by specialized benthos. However, scarce genomic resources severely limit our knowledge about the origin and adaptation of life in this unique ecosystem. Here, we present a genome of a deep-sea limpet Bathyacmaea lactea, a common species associated with the dominant mussel beds in cold seeps. We yielded 54.6 gigabases (Gb) of Nanopore reads and 77.9-Gb BGI-seq raw reads, respectively. Assembly harvested a 754.3-Mb genome for B. lactea, with 3,720 contigs and a contig N50 of 1.57 Mb, covering 94.3% of metazoan Benchmarking Universal Single-Copy Orthologs. In total, 23,574 protein-coding genes and 463.4 Mb of repetitive elements were identified. We analyzed the phylogenetic position, substitution rate, demographic history, and TE activity of B. lactea. We also identified 80 expanded gene families and 87 rapidly evolving Gene Ontology categories in the B. lactea genome. Many of these genes were associated with heterocyclic compound metabolism, membrane-bounded organelle, metal ion binding, and nitrogen and phosphorus metabolism. The high-quality assembly and in-depth characterization suggest the B. lactea genome will serve as an essential resource for understanding the origin and adaptation of life in the cold seeps.

scholarly journals Factors affecting de novo RNA synthesis and back-priming by the respiratory syncytial virus polymerase

Virology ◽  
2014 ◽  
Vol 462-463 ◽  
pp. 318-327 ◽  
Author(s):  
Sarah L. Noton ◽  
Waleed Aljabr ◽  
Julian A. Hiscox ◽  
David A. Matthews ◽  
Rachel Fearns
Keyword(s):  
Respiratory Syncytial Virus ◽  
Rna Synthesis ◽  
De Novo ◽  
Factors Affecting ◽  
Syncytial Virus

Glucocorticoid-regulated compartmentalization of cell surface-associated glycoproteins in rat hepatoma cells: evidence for an independent response that requires receptor function and de novo RNA synthesis

1987 ◽  
Vol 7 (4) ◽  
pp. 1508-1517
Author(s):  
O K Haffar ◽  
A K Vallerga ◽  
S A Marenda ◽  
H J Witchel ◽  
G L Firestone
Keyword(s):  
Cell Surface ◽  
Time Course ◽  
Rna Synthesis ◽  
De Novo ◽  
Receptor Occupancy ◽  
Receptor Function ◽  
Intracellular Fraction ◽  
Culture Cells ◽  
Viral Glycoproteins ◽  
Rat Hepatoma

The role of glucocorticoid hormones in the compartmentalization of cell surface-associated mouse mammary tumor virus (MMTV) glycoproteins was examined in M1.54, a cloned line of MMTV-infected rat hepatoma tissue culture cells. The expression of cellular [2-3H]mannose-labeled and cell surface 125I-labeled MMTV glycoproteins was examined throughout a time course of exposure to dexamethasone, a synthetic glucocorticoid. Posttranslational localization of cell surface MMTV glycoproteins required 6 h of exposure to hormone and occurred approximately 4 h after their initial production in an intracellular fraction. This regulated localization to the cell surface correlated with glucocorticoid receptor occupancy and was inhibited by exposure to RU 38486, a powerful antagonist of glucocorticoid-mediated responses. Cell surface immunoprecipitation demonstrated that actinomycin D, an inhibitor of de novo RNA synthesis, prevented regulated expression of cell surface viral glycoproteins, suggesting that newly synthesized cellular components mediate this process. The localization of cell surface MMTV glycoproteins appeared normal in a transcriptional variant (CR1) that produces basal levels of MMTV RNA and glycoprotein precursors in the presence of dexamethasone. Thus, regulated compartmentalization of viral glycoproteins is not an obligate consequence of a critical precursor concentration. Taken together, our results suggest that posttranslational trafficking of cell surface-destined MMTV glycoproteins resulted from an independent glucocorticoid hormone response that required receptor function and de novo RNA synthesis.

scholarly journals De Novo Initiation of RNA Synthesis by the RNA-Dependent RNA Polymerase (NS5B) of Hepatitis C Virus

2000 ◽  
Vol 74 (2) ◽  
pp. 851-863 ◽  
Author(s):  
Guangxiang Luo ◽  
Robert K. Hamatake ◽  
Danielle M. Mathis ◽  
Jason Racela ◽  
Karen L. Rigat ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Polymerase ◽  
Rna Synthesis ◽  
De Novo ◽  
Oligonucleotide Primer ◽  
Transferase Activity ◽  
Rdrp Activity ◽  
Rna Dependent Rna Polymerase ◽  
Hcv Ns5b

ABSTRACT Hepatitis C virus (HCV) NS5B protein possesses an RNA-dependent RNA polymerase (RdRp) activity, a major function responsible for replication of the viral RNA genome. To further characterize the RdRp activity, NS5B proteins were expressed from recombinant baculoviruses, purified to near homogeneity, and examined for their ability to synthesize RNA in vitro. As a result, a highly active NS5B RdRp (1b-42), which contains an 18-amino acid C-terminal truncation resulting from a newly created stop codon, was identified among a number of independent isolates. The RdRp activity of the truncated NS5B is comparable to the activity of the full-length protein and is 20 times higher in the presence of Mn2+ than in the presence of Mg2+. When a 384-nucleotide RNA was used as the template, two major RNA products were synthesized by 1b-42. One is a complementary RNA identical in size to the input RNA template (monomer), while the other is a hairpin dimer RNA synthesized by a “copy-back” mechanism. Substantial evidence derived from several experiments demonstrated that the RNA monomer was synthesized through de novo initiation by NS5B rather than by a terminal transferase activity. Synthesis of the RNA monomer requires all four ribonucleotides. The RNA monomer product was verified to be the result of de novo RNA synthesis, as two expected RNA products were generated from monomer RNA by RNase H digestion. In addition, modification of the RNA template by the addition of the chain terminator cordycepin at the 3′ end did not affect synthesis of the RNA monomer but eliminated synthesis of the self-priming hairpin dimer RNA. Moreover, synthesis of RNA on poly(C) and poly(U) homopolymer templates by 1b-42 NS5B did not require the oligonucleotide primer at high concentrations (≥50 μM) of GTP and ATP, further supporting a de novo initiation mechanism. These findings suggest that HCV NS5B is able to initiate RNA synthesis de novo.

Purine metabolism in Neisseria gonorrhoeae: the requirement for hypoxanthine

1980 ◽  
Vol 26 (1) ◽  
pp. 13-20 ◽  
Author(s):  
Stephen A. Morse ◽  
Lynne Bartenstein
Keyword(s):  
Neisseria Gonorrhoeae ◽  
Growth Medium ◽  
De Novo ◽  
Defined Medium ◽  
Purine Metabolism ◽  
Purine Biosynthesis ◽  
Free Medium ◽  
De Novo Purine Biosynthesis ◽  
Reduced Rate

Strains isolated from disseminated gonococcal infections often require hypoxanthine for growth. The biochemical bases for the requirement for hypoxanthine in strains isolated from both disseminated (Ile−Val−Arg−Hyx−Ura−phenotype) and non-disseminated (Hyx−phenotype) infections were compared. The requirement for hypoxanthine was dependent upon the composition of the growth medium. In a complete defined medium, hypoxanthine was replaced by a mixture of adenine and guanine but not by either purine alone. The addition of adenine alone inhibited gonococcal growth. This inhibition was reversed by the addition of guanine and most likely resulted from an inhibition of de novo purine biosynthesis. In a histidine-free medium, adenine replaced the hypoxanthine requirement in Ile−Val−Arg−Hyx−Ura− strains. Adenine did not replace the hypoxanthine requirement in Hyx− strains. The Ile−Val−Arg−Hyx−Ura− strains exhibited a markedly reduced rate of de novo purine biosynthesis while Hyx− strains were blocked in this pathway. In vivo concentrations of purines are important factors which may limit the intracellular or extracellular growth of these strains.

scholarly journals Cisplatin-Resistance in Oral Squamous Cell Carcinoma: Regulation by Tumor Cell-Derived Extracellular Vesicles

Cancers ◽  
2019 ◽  
Vol 11 (8) ◽  
pp. 1166 ◽  
Author(s):  
Xin-Hui Khoo ◽  
Ian C. Paterson ◽  
Bey-Hing Goh ◽  
Wai-Leng Lee
Keyword(s):  
Drug Resistance ◽  
Squamous Cell Carcinoma ◽  
Oral Squamous Cell Carcinoma ◽  
Cell Carcinoma ◽  
Extracellular Vesicles ◽  
Squamous Cell ◽  
Metal Ion ◽  
De Novo ◽  
Protein Profile ◽  
Severe Problem

Drug resistance remains a severe problem in most chemotherapy regimes. Recently, it has been suggested that cancer cell-derived extracellular vesicles (EVs) could mediate drug resistance. In this study, the role of EVs in mediating the response of oral squamous cell carcinoma (OSCC) cells to cisplatin was investigated. We isolated and characterized EVs from OSCC cell lines showing differential sensitivities to cisplatin. Increased EV production was observed in both de novo (H314) and adaptive (H103/cisD2) resistant lines compared to sensitive H103 cells. The protein profiles of these EVs were then analyzed. Differences in the proteome of EVs secreted by H103 and H103/cisD2 indicated that adaptation to cisplatin treatment caused significant changes in the secreted nanovesicles. Intriguingly, both resistant H103/cisD2 and H314 cells shared a highly similar EV protein profile including downregulation of the metal ion transporter, ATP1B3, in the EVs implicating altered drug delivery. ICP-MS analysis revealed that less cisplatin accumulated in the resistant cells, but higher levels were detected in their EVs. Therefore, we inhibited EV secretion from the cells using a proton pump inhibitor and observed an increased drug sensitivity in cisplatin-resistant H314 cells. This finding suggests that control of EV secretion could be a potential strategy to enhance the efficacy of cancer treatment.

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