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 Antibacterial and Anti-Inflammatory Activity of an Antimicrobial Peptide Synthesized with D Amino Acids

Antibiotics ◽  
2020 ◽  
Vol 9 (12) ◽  
pp. 840
Author(s):  
Jlenia Brunetti ◽  
Veronica Carnicelli ◽  
Alessia Ponzi ◽  
Antonio Di Giulio ◽  
Anna Rita Lizzi ◽  
...  
Keyword(s):  
Amino Acids ◽  
Low Frequency ◽  
Lipoteichoic Acid ◽  
Isomeric Form ◽  
Multiresistant Pathogens ◽  
Gram Negative ◽  
Anti Inflammatory ◽  
Strong Candidate

The peptide SET-M33 is a molecule synthesized in tetra-branched form which is being developed as a new antibiotic against Gram-negative bacteria. Its isomeric form with D amino acids instead of the L version (SET-M33D) is also able to kill Gram-positive bacteria because of its higher resistance to bacterial proteases (Falciani et al., PLoS ONE, 2012, 7, e46259). Here we report the strong in vitro activity of SET-M33D (MIC range 0.7–6.0 µM) against multiresistant pathogens of clinical interest, including Gram-positives Staphylococcus aureus, Staphylococcus saprophyticus, and Enterococcus faecalis, and various Gram-negative enterobacteriaceae. SET-M33D antibacterial activity is also confirmed in vivo against a MRSA strain of S. aureus with doses perfectly compatible with clinical use (5 and 2.5 mg/Kg). Moreover, SET-M33D strongly neutralized lipopolysaccharide (LPS) and lipoteichoic acid (LTA), thus exerting a strong anti-inflammatory effect, reducing expression of cytokines, enzymes, and transcription factors (TNF-α, IL6, COX-2, KC, MIP-1, IP10, iNOS, NF-κB) involved in the onset and evolution of the inflammatory process. These results, along with in vitro and in vivo toxicity data and the low frequency of resistance selection reported here, make SET-M33D a strong candidate for the development of a new broad spectrum antibiotic.

scholarly journals Expression of a dominant allele of human ARF1 inhibits membrane traffic in vivo

1994 ◽  
Vol 124 (3) ◽  
pp. 289-300 ◽  
Author(s):  
CJ Zhang ◽  
AG Rosenwald ◽  
MC Willingham ◽  
S Skuntz ◽  
J Clark ◽  
...  
Keyword(s):  
Golgi Apparatus ◽  
Rat Kidney ◽  
Brefeldin A ◽  
Membrane Traffic ◽  
Immunoblot Analysis ◽  
In Vitro Assays ◽  
Wild Type ◽  
Discernible Effect

ADP-ribosylation factor (ARF) proteins and inhibitory peptides derived from ARFs have demonstrated activities in a number of in vitro assays that measure ER-to-Golgi and intra-Golgi transport and endosome fusion. To better understand the roles of ARF proteins in vivo, stable cell lines were obtained from normal rat kidney (NRK) cells transfected with either wild-type or a dominant activating allele ([Q71L]) of the human ARF1 gene under the control of the interferon-inducible mouse Mx1 promoter. Upon addition of interferon, expression of ARF1 proteins increased with a half-time of 7-8 h, as determined by immunoblot analysis. Induction of mutant ARF1, but not wild-type ARF1, led to an inhibition of protein secretion with kinetics similar to that observed for induction of protein expression. Examination of the Golgi apparatus and the ER by indirect immunofluorescence or transmission electron microscopy revealed that expression of low levels of mutant ARF1 protein correlated with a dramatic increase in vesiculation of the Golgi apparatus and expansion of the ER lumen, while expression of substantially higher levels of wild-type ARF1 had no discernible effect. Endocytosis was also inhibited by expression of mutant ARF1, but not by the wild-type protein. Finally, the expression of [Q71L]ARF1, but not wild-type ARF1, antagonized the actions of brefeldin A, as determined by the delayed loss of ARF and beta-COP from Golgi membranes and disruption of the Golgi apparatus. General models for the actions of ARF1 in membrane traffic events are discussed.

scholarly journals Mutagenesis of solvent-exposed amino acids in Photinus pyralis luciferase improves thermostability and pH-tolerance

2006 ◽  
Vol 397 (2) ◽  
pp. 305-312 ◽  
Author(s):  
G. H. Erica Law ◽  
Olga A. Gandelman ◽  
Laurence C. Tisi ◽  
Christopher R. Lowe ◽  
James A. H. Murray
Keyword(s):  
Amino Acids ◽  
Specific Activity ◽  
Green Light ◽  
Firefly Luciferase ◽  
Wild Type ◽  
Photinus Pyralis ◽  
Wild Type Enzyme ◽  
Ph Tolerance

Firefly luciferase catalyses a two-step reaction, using ATP-Mg2+, firefly luciferin and molecular oxygen as substrates, leading to the efficient emission of yellow–green light. We report the identification of novel luciferase mutants which combine improved pH-tolerance and thermostability and that retain the specific activity of the wild-type enzyme. These were identified by the mutagenesis of solvent-exposed non-conserved hydrophobic amino acids to hydrophilic residues in Photinus pyralis firefly luciferase followed by in vivo activity screening. Mutants F14R, L35Q, V182K, I232K and F465R were found to be the preferred substitutions at the respective positions. The effects of these amino acid replacements are additive, since combination of the five substitutions produced an enzyme with greatly improved pH-tolerance and stability up to 45 °C. All mutants, including the mutant with all five substitutions, showed neither a decrease in specific activity relative to the recombinant wild-type enzyme, nor any substantial differences in kinetic constants. It is envisaged that the combined mutant will be superior to wild-type luciferase for many in vitro and in vivo applications.

scholarly journals Evaluation of Anti-inflammatory, Anti-pyretic, Analgesic, and Hepatoprotective Properties of Terminalia macroptera

2020 ◽  
Vol 07 (02) ◽  
pp. e58-e67
Author(s):  
Mahamane Haïdara ◽  
Adama Dénou ◽  
Mohamed Haddad ◽  
Aïssata Camara ◽  
Korotoumou Traoré ◽  
...  
Keyword(s):  
Traditional Medicine ◽  
High Resolution Mass Spectrometry ◽  
Biological Activities ◽  
Traditional Healers ◽  
Potential Candidate ◽  
Murine Models ◽  
Leaf Extracts ◽  
Anti Inflammatory

AbstractIn Mali, improved traditional medicines [“Médicaments Traditionnels Améliorés”] are prepared from traditionally used medicinal plants. Recently, the Department of Traditional Medicine has identified Terminalia macroptera Guill. & Perr. (Combretaceae) as a potential candidate for an improved traditional medicine. T. macroptera is a West African medicinal plant used in Mali against various health disorders, with more than 30 different indications mentioned by traditional healers, including hepatitis, gonorrhea, fever, pain relief, and various infectious diseases (Helicobacter pylori-associated diseases). To date, validation of most of the biological activities of has been mainly carried out in vitro, except for antimalarial activities. In this study, the potential anti-inflammatory, antipyretic, analgesic, and hepatoprotective properties of T. macroptera were investigated in different murine models. Administration of T. macroptera ethanolic root and leaf extracts in rats significantly reduced pyrexia, pain, inflammation, and hepatic marker enzymes such as alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase in the different murine models used (p<0.05). A phytochemical screening of T. macroptera revealed the presence of tannins, flavonoids, saponins, anthracene derivatives, sterols, triterpenes, and sugars in both leaf and root extracts as the main phytochemical compounds. This was confirmed by qualitative analysis, liquid chromatography coupled with high-resolution mass spectrometry. T. macroptera extracts demonstrated interesting in vivo antipyretic, analgesic, anti-inflammatory, and hepatoprotective activities. Therefore, T. macroptera should be proposed and further evaluated as a potential improved traditional medicine for the treatment of liver-related disorders and for the relief of pain and fever.

scholarly journals A potent bispecific nanobody protects hACE2 mice against SARS-CoV-2 infection via intranasal administration

2021 ◽  
Author(s):  
Xilin Wu ◽  
Lin Cheng ◽  
Ming Fu ◽  
Bilian Huang ◽  
Linjing Zhu ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Intranasal Administration ◽  
List Type ◽  
Potential Candidate ◽  
Intranasal Delivery ◽  
Wild Type ◽  
Viral Inhibition ◽  
Live Virus

AbstractThe dramatically expanding COVID-19 needs multiple effective countermeasures. Neutralizing antibodies are a potential therapeutic strategy for treating COVID-19. A number of neutralizing nanobodies (Nbs) were reported for their in vitro activities. However, in vivo protection of these nanobodies was not reported in animal models. In the current report, we characterized several RBD-specific Nbs isolated from a screen of an Nb library derived from an alpaca immunized with SARS-CoV-2 spike glycoprotein (S); among them, three Nbs exhibited picomolar potency against SARS-CoV-2 live virus, pseudotyped viruses, and 15 circulating SARS-CoV-2 variants. To improve the efficacy, various configurations of Nbs were engineered. Nb15-NbH-Nb15, a novel trimer constituted of three Nbs, was constructed to be bispecific for human serum albumin (HSA) and RBD of SARS-CoV-2. Nb15-NbH-Nb15 exhibited sub-ng/ml neutralization potency against the wild-type and currently circulating variants of SARS-CoV-2 with a long half-life in vivo. In addition, we showed that intranasal administration of Nb15-NbH-Nb15 provided 100% protection for both prophylactic and therapeutic purposes against SARS-CoV-2 infection in transgenic hACE2 mice. Nb15-NbH-Nb15 is a potential candidate for both prevention and treatment of SARS-CoV-2 through respiratory administration.One sentence summaryNb15-NbH-Nb15, with a novel heterotrimeric bispecific configuration, exhibited potent and broad neutralization potency against SARS-CoV-2 in vitro and provided in vivo protection against SARS-CoV-2 infection in hACE2 transgenic mice via intranasal delivery.Graphical abstract:HighlightsWe described a novel heterotrimeric configuration of Nb-NbH-Nb (Nb15-NbH-Nb15) that exhibited improved viral inhibition and stability.Nb15-NbH-Nb15 provides ultrahigh neutralization potency against SARS-CoV-2 wild type and 18 mutant variants, including the current circulating variants of D614G and N501Y predominantly in the UK and South Africa.It is the first to demonstrate the Nbs efficacy in preventing and treating SARS-CoV-2 infection in hACE2 transgenic mice via intranasal delivery.

scholarly journals Anti-Inflammatory, Antipyretic, and Analgesic Properties of Potamogeton perfoliatus Extract: In Vitro and In Vivo Study

Molecules ◽  
2021 ◽  
Vol 26 (16) ◽  
pp. 4826
Author(s):  
Samar Rezq ◽  
Mona F. Mahmoud ◽  
Assem M. El-Shazly ◽  
Mohamed A. El Raey ◽  
Mansour Sobeh
Keyword(s):  
Oxidative Stress ◽  
Acetic Acid ◽  
Biological Activities ◽  
Opioid Analgesic ◽  
Potential Candidate ◽  
Inflammatory Models ◽  
Potamogeton Perfoliatus ◽  
Anti Inflammatory

Natural antioxidants, especially those of plant origins, have shown a plethora of biological activities with substantial economic value, as they can be extracted from agro-wastes and/or under exploited plant species. The perennial hydrophyte, Potamogeton perfoliatus, has been used traditionally to treat several health disorders; however, little is known about its biological and its medicinal effects. Here, we used an integrated in vitro and in vivo framework to examine the potential effect of P. perfoliatus on oxidative stress, nociception, inflammatory models, and brewer’s yeast-induced pyrexia in mice. Our results suggested a consistent in vitro inhibition of three enzymes, namely 5-lipoxygenase, cyclooxygenases 1 and 2 (COX-1 and COX-2), as well as a potent antioxidant effect. These results were confirmed in vivo where the studied extract attenuated carrageenan-induced paw edema, carrageenan-induced leukocyte migration into the peritoneal cavity by 25, 44 and 64% at 200, 400 and 600 mg/kg, p.o., respectively. Moreover, the extract decreased acetic acid-induced vascular permeability by 45% at 600 mg/kg, p.o., and chemical hyperalgesia in mice by 86% by 400 mg/kg, p.o., in acetic acid-induced writhing assay. The extract (400 mg/kg) showed a longer response latency at the 3 h time point (2.5 fold of the control) similar to the nalbuphine, the standard opioid analgesic. Additionally, pronounced antipyretic effects were observed at 600 mg/kg, comparable to paracetamol. Using LC-MS/MS, we identified 15 secondary metabolites that most likely contributed to the obtained biological activities. Altogether, our findings indicate that P. perfoliatus has anti-inflammatory, antioxidant, analgesic and antipyretic effects, thus supporting its traditional use and promoting its valorization as a potential candidate in treating oxidative stress-associated diseases.

scholarly journals A Naturally Occurring Deletion in FliE from Salmonella enterica Serovar Dublin Results in an Aflagellate Phenotype and Defective Proinflammatory Properties

2017 ◽  
Vol 86 (1) ◽  
Author(s):  
Sebastián Sasías ◽  
Adriana Martínez-Sanguiné ◽  
Laura Betancor ◽  
Arací Martínez ◽  
Bruno D'Alessandro ◽  
...  
Keyword(s):  
Amino Acids ◽  
Dna Sequences ◽  
High Frequency ◽  
Salmonella Enterica ◽  
Wild Type ◽  
Content Type ◽  
Naturally Occurring ◽  
Salmonella Enterica Serovar Dublin

ABSTRACTSalmonella entericaserovar Dublin is adapted to cattle but is able to infect humans with high invasiveness. An acute inflammatory response at the intestine helps to preventSalmonelladissemination to systemic sites. Flagella contribute to this response by providing motility and FliC-mediated signaling through pattern recognition receptors. In a previous work, we reported a high frequency (11 out of 25) ofS. Dublin isolates lacking flagella in a collection obtained from humans and cattle. The aflagellate strains were impaired in their proinflammatory propertiesin vitroandin vivo. The aim of this work was to elucidate the underlying cause of the absence of flagella inS. Dublin isolates. We report here that class 3 flagellar genes are repressed in the human aflagellate isolates, due to impaired secretion of FliA anti-sigma factor FlgM. This phenotype is due to an in-frame 42-nucleotide deletion in thefliEgene, which codes for a protein located in the flagellar basal body. The deletion is predicted to produce a protein lacking amino acids 18 to 31. The aflagellate phenotype was highly stable; revertants were obtained only whenfliAwas artificially overexpressed combined with several successive passages in motility agar. DNA sequence analysis revealed that motile revertants resulted from duplications of DNA sequences infliEadjacent to the deleted region. These duplications produced a FliE protein of similar length to the wild type and demonstrate that amino acids 18 to 31 of FliE are not essential. The same deletion was detected inS. Dublin isolates obtained from cattle, indicating that this mutation circulates in nature.

In Vivo and In Vitro Anti-inflammatory Activities of Neoandrographolide

2007 ◽  
Vol 35 (02) ◽  
pp. 317-328 ◽  
Author(s):  
Jun Liu ◽  
Zheng-Tao Wang ◽  
Li-Li Ji
Keyword(s):  
Oral Administration ◽  
Potential Candidate ◽  
Necrosis Factor Alpha ◽  
Tnf Α ◽  
Factor Alpha ◽  
Anti Inflammatory ◽  
Necrosis Factor ◽  
Dimethyl Benzene

Neoandrographolide, one of the principal diterpene lactones, isolated from a medicinal herb Andrographis paniculata Nees, was tested in vivo and in vitro for its anti-inflammatory activities and mechanism. Oral administration of neoandrographolide (150 mg/kg) significantly suppressed ear edema induced by dimethyl benzene in mice. Oral administration of neoandrographolide (100–150 mg/kg) also reduced the increase in vascular permeability induced by acetic acid in mice. In vitro studies were performed using the macrophage cell line RAW264.7 to study the effect of neoandrographolide on suppressing phorbol-12-myristate-13-acetate (PMA)-stimulated respiratory bursts and lipopolysaccharide (LPS)-induced production of nitric oxide (NO) and tumor necrosis factor-alpha (TNF-α). Respiratory bursts were quantified by chemiluminescence (CL) measurements.Results showed that neoandrographolide suppressed PMA-stimulated respiratory bursts dose-dependently from 30 μM to 150 μM. Neoandrographolide also inhibited NO and TNF-α production in LPS-induced macrophages, contributing to the anti-inflammatory activity of A. paniculata. These results indicate that neoandrographolide possesses significant anti-inflammatory effects, which implies that it would be one of the major contributing components to participate in the anti-inflammatory effect of A. paniculata. and a potential candidate for further clinical trial.

scholarly journals Overexpression of Wild-Type Aspartokinase Increases l-Lysine Production in the Thermotolerant Methylotrophic Bacterium Bacillus methanolicus

2008 ◽  
Vol 75 (3) ◽  
pp. 652-661 ◽  
Author(s):  
�yvind M. Jakobsen ◽  
Trygve Brautaset ◽  
Kristin F. Degnes ◽  
Tonje M. B. Heggeset ◽  
Simone Balzer ◽  
...  
Keyword(s):  
Amino Acids ◽  
Methylotrophic Bacterium ◽  
Wild Type ◽  
Lysine Production ◽  
Bacillus Methanolicus ◽  
Semialdehyde Dehydrogenase ◽  
Aspartate Pathway ◽  
The Impact

ABSTRACT Aspartokinase (AK) controls the carbon flow into the aspartate pathway for the biosynthesis of the amino acids l-methionine, l-threonine, l-isoleucine, and l-lysine. We report here the cloning of four genes (asd, encoding aspartate semialdehyde dehydrogenase; dapA, encoding dihydrodipicolinate synthase; dapG, encoding AKI; and yclM, encoding AKIII) of the aspartate pathway in Bacillus methanolicus MGA3. Together with the known AKII gene lysC, dapG and yclM form a set of three AK genes in this organism. Overexpression of dapG, lysC, and yclM increased l-lysine production in wild-type B. methanolicus strain MGA3 2-, 10-, and 60-fold (corresponding to 11 g/liter), respectively, without negatively affecting the specific growth rate. The production levels of l-methionine (less than 0.5 g/liter) and l-threonine (less than 0.1 g/liter) were low in all recombinant strains. The AK proteins were purified, and biochemical analyses demonstrated that they have similar V max values (between 47 and 58 μmol/min/mg protein) and Km values for l-aspartate (between 1.9 and 5.0 mM). AKI and AKII were allosterically inhibited by meso-diaminopimelate (50% inhibitory concentration [IC50], 0.1 mM) and by l-lysine (IC50, 0.3 mM), respectively. AKIII was inhibited by l-threonine (IC50, 4 mM) and by l-lysine (IC50, 5 mM), and this enzyme was synergistically inhibited in the presence of both of these amino acids at low concentrations. The correlation between the impact on l-lysine production in vivo and the biochemical properties in vitro of the individual AK proteins is discussed. This is the first example of improving l-lysine production by metabolic engineering of B. methanolicus and also the first documentation of considerably increasing l-lysine production by overexpression of a wild-type AK.

scholarly journals The carboxyl terminus of the human cytomegalovirus UL37 immediate-early glycoprotein is conserved in primary strains and is important for transactivation

2001 ◽  
Vol 82 (7) ◽  
pp. 1569-1579 ◽  
Author(s):  
Wail A. Hayajneh ◽  
Despina G. Contopoulos-Ioannidis ◽  
Marci M. Lesperance ◽  
Ana M. Venegas ◽  
Anamaris M. Colberg-Poley
Keyword(s):  
Amino Acids ◽  
Human Cytomegalovirus ◽  
Immediate Early ◽  
Carboxyl Terminus ◽  
Wild Type ◽  
Reading Frame ◽  
Cytosolic Domains ◽  
Carboxyl Terminal

The human cytomegalovirus (HCMV) UL37 exon 3 (UL37x3) open reading frame (ORF) encodes the carboxyl termini of two immediate-early glycoproteins (gpUL37 and gpUL37M). UL37x3 homologous sequences are not required for mouse cytomegalovirus (MCMV) growth in vitro; yet, they are important for MCMV growth and pathogenesis in vivo. Similarly, UL37x3 sequences are dispensable for HCMV growth in culture, but their requirement for HCMV growth in vivo is not known. To determine this requirement, we directly sequenced the complete UL37x3 gene in multiple HCMV primary strains. A total of 63 of the 310 amino acids in the UL37x3 ORF differ non-conservatively in one or more HCMV primary strains. The HCMV UL37x3 genetic diversity is non-random: the N-glycosylation (46/186 aa) and basic (9/15 aa) domains have the highest proportion of non-conservative variant amino acids. Nonetheless, most (15/17 signals) of the N-glycosylation signals are retained in all HCMV primary strains. Moreover, new N-glycosylation signals are encoded by 5/20 primary strains. In sharp contrast, the UL37x3 transmembrane (TM) ORF completely lacks diversity in all 20 HCMV sequenced primary strains, and only 1 of 28 cytosolic tail residues differs non-conservatively. To test the functional significance of the conserved carboxyl terminus, gpUL37 mutants lacking the TM and/or cytosolic tail were tested for transactivating activity. The gpUL37 carboxyl-terminal mutants are partially defective in hsp70 promoter transactivation even though they trafficked similarly to the wild-type protein into the endoplasmic reticulum and to mitochondria. From these results, we conclude that N-glycosylated gpUL37, particularly its TM and cytosolic domains, is important for HCMV growth in humans.

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