Regulatory Functions of Serine-46-Phosphorylated HPr in Lactococcus lactis

ABSTRACT In most low-G+C gram-positive bacteria, the phosphoryl carrier protein HPr of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) becomes phosphorylated at Ser-46. This ATP-dependent reaction is catalyzed by the bifunctional HPr kinase/P-Ser-HPr phosphatase. We found that serine-phosphorylated HPr (P-Ser-HPr) of Lactococcus lactis participates not only in carbon catabolite repression of an operon encoding a β-glucoside-specific EII and a 6-P-β-glucosidase but also in inducer exclusion of the non-PTS carbohydrates maltose and ribose. In a wild-type strain, transport of these non-PTS carbohydrates is strongly inhibited by the presence of glucose, whereas in a ptsH1 mutant, in which Ser-46 of HPr is replaced with an alanine, glucose had lost its inhibitory effect. In vitro experiments carried out with L. lactis vesicles had suggested that P-Ser-HPr is also implicated in inducer expulsion of nonmetabolizable homologues of PTS sugars, such as methylβ-d-thiogalactoside (TMG) and 2-deoxy-d-glucose (2-DG). In vivo experiments with theptsH1 mutant established that P-Ser-HPr is not necessary for inducer expulsion. Glucose-activated 2-DG expulsion occurred at similar rates in wild-type and ptsH1 mutant strains, whereas TMG expulsion was slowed in the ptsH1 mutant. It therefore seems that P-Ser-HPr is not essential for inducer expulsion but that in certain cases it can play an indirect role in this regulatory process.

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Synthesis and Characterization of Polyvinylpyrrolidone-Modified ZnO Quantum Dots and Their In Vitro Photodynamic Tumor Suppressive Action

International Journal of Molecular Sciences ◽

10.3390/ijms22158106 ◽

2021 ◽

Vol 22 (15) ◽

pp. 8106

Author(s):

Tianming Song ◽

Yawei Qu ◽

Zhe Ren ◽

Shuang Yu ◽

Mingjian Sun ◽

...

Keyword(s):

Quantum Dots ◽

Photodynamic Therapy ◽

Inhibitory Effect ◽

Therapeutic Effects ◽

In Vivo Experiments ◽

Potential Applications ◽

Zno Quantum Dots ◽

Zno Qds

Despite the numerous available treatments for cancer, many patients succumb to side effects and reoccurrence. Zinc oxide (ZnO) quantum dots (QDs) are inexpensive inorganic nanomaterials with potential applications in photodynamic therapy. To verify the photoluminescence of ZnO QDs and determine their inhibitory effect on tumors, we synthesized and characterized ZnO QDs modified with polyvinylpyrrolidone. The photoluminescent properties and reactive oxygen species levels of these ZnO/PVP QDs were also measured. Finally, in vitro and in vivo experiments were performed to test their photodynamic therapeutic effects in SW480 cancer cells and female nude mice. Our results indicate that the ZnO QDs had good photoluminescence and exerted an obvious inhibitory effect on SW480 tumor cells. These findings illustrate the potential applications of ZnO QDs in the fields of photoluminescence and photodynamic therapy.

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PTS 50: Past, Present and Future, or Diauxie Revisited

Journal of Molecular Microbiology and Biotechnology ◽

10.1159/000369809 ◽

2015 ◽

Vol 25 (2-3) ◽

pp. 79-93 ◽

Cited By ~ 9

Author(s):

Joseph W. Lengeler

Keyword(s):

Catabolite Repression ◽

Biological Significance ◽

Cellular Growth ◽

Gram Positive ◽

Gram Negative ◽

Inducer Exclusion ◽

Gram Positive Bacteria ◽

Cellular Control

Past: The title ‘PTS 50 or The PTS after 50 years' relies on the first description in 1964 of the phosphoenolpyruvate-dependent carbohydrate:phosphotransferase system (PTS) by Kundig, Gosh and Roseman [Proc Natl Acad Sci USA 1964;52:1067-1074]. The system comprised proteins named Enzyme I, HPr and Enzymes II, as part of a novel PTS for carbohydrates in Gram-negative and Gram-positive bacteria, whose ‘biological significance remained unclear'. In contrast, studies which would eventually lead to the discovery of the central role of the PTS in bacterial metabolism had been published since before 1942. They are primarily linked to names like Epps and Gale, J. Monod, Cohn and Horibata, and B. Magasanik, and to phenomena like ‘glucose effects', ‘diauxie', ‘catabolite repression' and carbohydrate transport. Present: The pioneering work from Roseman's group initiated a flood of publications. The extraordinary progress from 1964 to this day in the qualitative and in vitro description of the genes and enzymes of the PTS, and of its multiple roles in global cellular control through ‘inducer exclusion', gene induction and ‘catabolite repression', in cellular growth, in cell differentiation and in chemotaxis, as well as the differences of its functions between Gram-positive and Gram-negative bacteria, was one theme of the meeting and will not be treated in detail here. Future: At the 1988 Paris meeting entitled ‘The PTS after 25 years', Saul Roseman predicted that ‘we must describe these interactions [of the PTS components] in a quantitative way [under] in vivo conditions'. I will present some results obtained by our group during recent years on the old phenomenon of diauxie by means of very fast and quantitative tests, measured in vivo, and obtained from cultures of isogenic mutant strains growing under chemostat conditions. The results begin to hint at the problems relating to future PTS research, but also to the ‘true science' of Roseman.

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Tenovin-6 impairs autophagy by inhibiting autophagic flux

Cell Death and Disease ◽

10.1038/cddis.2017.25 ◽

2017 ◽

Vol 8 (2) ◽

pp. e2608-e2608 ◽

Cited By ~ 12

Author(s):

Hongfeng Yuan ◽

Brandon Tan ◽

Shou-Jiang Gao

Keyword(s):

Cell Types ◽

Inhibitory Effect ◽

Lymphocytic Leukemia ◽

Autophagic Flux ◽

Dependent Manner ◽

Autophagy Pathway ◽

Sarcoma Cells ◽

Regulatory Functions

Abstract Tenovin-6 has attracted significant interest because it activates p53 and inhibits sirtuins. It has anti-neoplastic effects on multiple hematopoietic malignancies and solid tumors in both in vitro and in vivo studies. Tenovin-6 was recently shown to impair the autophagy pathway in chronic lymphocytic leukemia cells and pediatric soft tissue sarcoma cells. However, whether tenovin-6 has a general inhibitory effect on autophagy and whether there is any involvement with SIRT1 and p53, both of which are regulators of the autophagy pathway, remain unclear. In this study, we have demonstrated that tenovin-6 increases microtubule-associated protein 1 light chain 3 (LC3-II) level in diverse cell types in a time- and dose-dependent manner. Mechanistically, the increase of LC3-II by tenovin-6 is caused by inhibition of the classical autophagy pathway via impairing lysosomal function without affecting the fusion between autophagosomes and lysosomes. Furthermore, we have revealed that tenovin-6 activation of p53 is cell type dependent, and tenovin-6 inhibition of autophagy is not dependent on its regulatory functions on p53 and SIRT1. Our results have shown that tenovin-6 is a potent autophagy inhibitor, and raised the precaution in interpreting results where tenovin-6 is used as an inhibitor of SIRT1.

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Asthenozoospermia in Mice with Targeted Deletion of the Sperm Mitochondrion-Associated Cysteine-Rich Protein (Smcp) Gene

Molecular and Cellular Biology ◽

10.1128/mcb.22.9.3046-3052.2002 ◽

2002 ◽

Vol 22 (9) ◽

pp. 3046-3052 ◽

Cited By ~ 74

Author(s):

Karim Nayernia ◽

Ibrahim M. Adham ◽

Elke Burkhardt-Göttges ◽

Jürgen Neesen ◽

Mandy Rieche ◽

...

Keyword(s):

Semen Analysis ◽

Computer Assisted ◽

Microscopical Examination ◽

Structural Protein ◽

Wild Type ◽

Vitro Fertilization ◽

In Vivo Experiments ◽

Cysteine Rich Protein

ABSTRACT The sperm mitochondria-associated cysteine-rich protein (SMCP) is a cysteine- and proline-rich structural protein that is closely associated with the keratinous capsules of sperm mitochondria in the mitochondrial sheath surrounding the outer dense fibers and axoneme. To investigate the function of SMCP, we generated mice with a targeted disruption of the gene Smcp by homologous recombination. Homozygous mutant males on a mixed genetic background (C57BL/6J × 129/Sv) are fully fertile, while they are infertile on the 129/Sv background, although spermatogenesis and mating are normal. Homozygous Smcp−/− female mice are fertile on both genetic backgrounds. Electron microscopical examination demonstrated normal structures of sperm head, mitochondria, and tail. In vivo experiments with sperm of Smcp−/− 129/Sv mice revealed that the migration of spermatozoa from the uterus into the oviduct is reduced. This result is supported by the observation that sperm motility as determined by the computer-assisted semen analysis system (CASA) is significantly affected as compared to wild-type spermatozoa. In vitro fertilization assays showed that Smcp-deficient spermatozoa are able to bind to the oocyte but that the number of fertilized eggs is reduced by more than threefold relative to the wild-type control. However, removal of the zona pellucida resulted in an unaffected sperm-egg fusion which was monitored by the presence of pronuclei and generation of blastocyts. These results indicate that the infertility of the male Smcp−/− mice on the 129/Sv background is due to reduced motility of the spermatozoa and decreased capability of the spermatozoa to penetrate oocytes.

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Intestinal P Glycoprotein Acts as a Natural Defense Mechanism against Listeria monocytogenes

Infection and Immunity ◽

10.1128/iai.72.7.3849-3854.2004 ◽

2004 ◽

Vol 72 (7) ◽

pp. 3849-3854 ◽

Cited By ~ 23

Author(s):

Brien L. Neudeck ◽

Jennifer M. Loeb ◽

Nancy G. Faith ◽

Charles J. Czuprynski

Keyword(s):

Listeria Monocytogenes ◽

Defense Mechanism ◽

Transepithelial Transport ◽

Bacterial Invasion ◽

Wild Type ◽

In Vivo Experiments ◽

Natural Defense ◽

P Glycoprotein

ABSTRACT Mechanisms by which the intestinal epithelium resists invasion by food-borne pathogens such as Listeria monocytogenes are an evolving area of research. Intestinal P glycoprotein is well known to limit the absorption of xenobiotics and is believed to act as a cytotoxic defense mechanism. The aim of this study was to determine if intestinal P glycoprotein is involved in host defense against L. monocytogenes. Caco-2 cells and a P-glycoprotein-overexpressing subclone (Caco-2/MDR) were employed in addition to mdr1a−/− mice and wild-type controls. In vitro invasion assays and in vivo experiments were employed to measure bacterial invasion and dissemination. In addition, L. monocytogenes proteins were labeled with [35S]methionine, and the transepithelial transport across Caco-2 monolayers was characterized in both directions. Overexpression of P glycoprotein in Caco-2/MDR cells led to increased resistance to L. monocytogenes invasion, whereas P-glycoprotein inhibition led to increased invasion. Flux of [35S]methionine-labeled L. monocytogenes proteins was significantly greater in the basolateral-to-apical direction than in the apical-to-basolateral direction, indicating dependence on an apically located efflux transporter. Moreover, inhibiting P glycoprotein reduced the basolateral-to-apical flux of the proteins. Early dissemination of L. monocytogenes from the gastrointestinal tract was significantly greater in the mdr1a−/− mice than in wild-type controls. Expression and function of intestinal P glycoprotein is an important determinant in resistance to early invasion of L. monocytogenes.

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Isolation Purification and Partial Characterization of Antisnake Venom Plant Peptide (BRS-P19) from Bauhinia rufescens (LAM FAM) Seed as Potential Alternative to Serum-Based Antivenin

Journal of Biotechnology Research ◽

10.32861/jbr.64.18.26 ◽

2020 ◽

pp. 18-26

Author(s):

I. Sani ◽

A.A. Umar ◽

S.A. Jiga ◽

F. Bello ◽

A. Abdulhamid ◽

...

Keyword(s):

Antioxidant Enzymes ◽

Research Work ◽

Gel Filtration ◽

Inhibitory Effect ◽

Untreated Group ◽

Control Group ◽

In Vivo Experiments ◽

Potential Alternative

Several studies have been reported on active peptides isolated from some medicinal plants, which were effective inhibitors against snake venom induced toxicities. Hence, the aim of this research work was to isolate, purify and characterize an antisnake venom plant peptide from Bauhinia rufescens seed that can serve as potential alternative to serum-based antivenins. B. rufescens seed was collected, duly identified, authenticated and processed. The peptide was isolated from the seed and purified using gel filtration chromatography and SDS-PAGE and then named as BRS-P19. Venom Phospholipase A2 (VPLA2) was used for the study and was isolated from Naja nigricollis venom. Albino mice of both sexes were used for in vivo experiments. They were divided into seven (7) groups of three (3) mice each. Group 1 served as normal control, group 2 were injected with VPLA2 only, group 3 and 4 were injected with VPLA2 then treated with BRS-P19 at doses of 0.2 and 0.4 mg/kg b.w. respectively, while mice in group 5 were injected with VPLA2 then treated with standard antivenin, group 6 and 7 were injected with VPLA2 followed by administration of ascorbic acid and α-tocopherol respectively. In all the groups, hepatic and renal levels of reactive oxygen species (ROS), lipid peroxidation (MDA) and activities of antioxidant enzymes were determined. The results showed that, the BRS-P19 has molecular weight of ~19kD. Its percentage in vitro inhibitory effect against VPLA2 was 91.85 ± 0.32%. For the in vivo study, the animals treated with 0.4 mg/kg b.w. of the BRS-P19 showed a significant (P<0.05) decrease in the hepatic and renal ROS and MDA levels when compared with the VPLA2 untreated group. But, the activities of the antioxidant enzymes in all the treated groups were significantly (P<0.05) increased by the BRS-P19 at 0.4 mg/kg b.w. when compared to the VPLA2 untreated group. Based on these findings, it has been established that, BRS-P19 has antisnake venom effect through inhibition of VPLA2 and antioxidant activity as the possible mechanisms of action.

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A new adenylyl cyclase, putative disease-resistance RPP13-like protein 3, participates in abscisic acid-mediated resistance to heat stress in maize

Journal of Experimental Botany ◽

10.1093/jxb/eraa431 ◽

2020 ◽

Cited By ~ 2

Author(s):

Hao Yang ◽

Yulong Zhao ◽

Ning Chen ◽

Yanpei Liu ◽

Shaoyu Yang ◽

...

Keyword(s):

Heat Stress ◽

Disease Resistance ◽

Cyclic Adenosine Monophosphate ◽

Adenosine Monophosphate ◽

Wild Type ◽

In Vivo Experiments ◽

In The Wild ◽

Shock Proteins

Abstract In plants, 3´,5´-cyclic adenosine monophosphate (cAMP) is an important second messenger with varied functions; however, only a few adenylyl cyclases (ACs) that synthesize cAMP have been identified. Moreover, the biological roles of ACs/cAMP in response to stress remain largely unclear. In this study, we used quantitative proteomics techniques to identify a maize heat-induced putative disease-resistance RPP13-like protein 3 (ZmRPP13-LK3), which has three conserved catalytic AC centres. The AC activity of ZmRPP13-LK3 was confirmed by in vitro enzyme activity analysis, in vivo RNAi experiments, and functional complementation in the E. coli cyaA mutant. ZmRPP13-LK3 is located in the mitochondria. The results of in vitro and in vivo experiments indicated that ZmRPP13-LK3 interacts with ZmABC2, a possible cAMP exporter. Under heat stress, the concentrations of ZmRPP13-LK3 and cAMP in the ABA-deficient mutant vp5 were significantly less than those in the wild-type, and treatment with ABA and an ABA inhibitor affected ZmRPP13-LK3 expression in the wild-type. Application of 8-Br-cAMP, a cAMP analogue, increased heat-induced expression of heat-shock proteins in wild-type plants and alleviated heat-activated oxidative stress. Taken together, our results indicate that ZmRPP13-LK3, a new AC, can catalyse ATP for the production of cAMP and may be involved in ABA-regulated heat resistance.

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Reverse Genetics of Escherichia coliGlycerol Kinase Allosteric Regulation and Glucose Control of Glycerol Utilization In Vivo

Journal of Bacteriology ◽

10.1128/jb.183.11.3336-3344.2001 ◽

2001 ◽

Vol 183 (11) ◽

pp. 3336-3344 ◽

Cited By ~ 36

Author(s):

C. Kay Holtman ◽

Aaron C. Pawlyk ◽

Norman D. Meadow ◽

Donald W. Pettigrew

Keyword(s):

Glucose Control ◽

Reverse Genetics ◽

Allosteric Regulation ◽

Glycerol Kinase ◽

Phosphotransferase System ◽

Inducer Exclusion ◽

Glycerol Utilization ◽

Fructose 1,6 Bisphosphate

ABSTRACT Reverse genetics is used to evaluate the roles in vivo of allosteric regulation of Escherichia coli glycerol kinase by the glucose-specific phosphocarrier of the phosphoenolpyruvate:glycose phosphotransferase system, IIAGlc (formerly known as IIIglc), and by fructose 1,6-bisphosphate. Roles have been postulated for these allosteric effectors in glucose control of both glycerol utilization and expression of the glpK gene. Genetics methods based on homologous recombination are used to place glpKalleles with known specific mutations into the chromosomal context of the glpK gene in three different genetic backgrounds. The alleles encode glycerol kinases with normal catalytic properties and specific alterations of allosteric regulatory properties, as determined by in vitro characterization of the purified enzymes. TheE. coli strains with these alleles display the glycerol kinase regulatory phenotypes that are expected on the basis of the in vitro characterizations. Strains with different glpRalleles are used to assess the relationships between allosteric regulation of glycerol kinase and specific repression in glucose control of the expression of the glpK gene. Results of these studies show that glucose control of glycerol utilization and glycerol kinase expression is not affected by the loss of IIAGlc inhibition of glycerol kinase. In contrast, fructose 1,6-bisphosphate inhibition of glycerol kinase is the dominant allosteric control mechanism, and glucose is unable to control glycerol utilization in its absence. Specific repression is not required for glucose control of glycerol utilization, and the relative roles of various mechanisms for glucose control (catabolite repression, specific repression, and inducer exclusion) are different for glycerol utilization than for lactose utilization.

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Why Does Escherichia coli Grow More Slowly on Glucosamine than on N-Acetylglucosamine? Effects of Enzyme Levels and Allosteric Activation of GlcN6P Deaminase (NagB) on Growth Rates

Journal of Bacteriology ◽

10.1128/jb.187.9.2974-2982.2005 ◽

2005 ◽

Vol 187 (9) ◽

pp. 2974-2982 ◽

Cited By ~ 47

Author(s):

Laura I. Álvarez-Añorve ◽

Mario L. Calcagno ◽

Jacqueline Plumbridge

Keyword(s):

Escherichia Coli ◽

Growth Rates ◽

Sugar Transport ◽

Point Mutations ◽

Sole Source ◽

Wild Type ◽

Phosphotransferase System ◽

Allosteric Activation

ABSTRACT Wild-type Escherichia coli grows more slowly on glucosamine (GlcN) than on N-acetylglucosamine (GlcNAc) as a sole source of carbon. Both sugars are transported by the phosphotransferase system, and their 6-phospho derivatives are produced. The subsequent catabolism of the sugars requires the allosteric enzyme glucosamine-6-phosphate (GlcN6P) deaminase, which is encoded by nagB, and degradation of GlcNAc also requires the nagA-encoded enzyme, N-acetylglucosamine-6-phosphate (GlcNAc6P) deacetylase. We investigated various factors which could affect growth on GlcN and GlcNAc, including the rate of GlcN uptake, the level of induction of the nag operon, and differential allosteric activation of GlcN6P deaminase. We found that for strains carrying a wild-type deaminase (nagB) gene, increasing the level of the NagB protein or the rate of GlcN uptake increased the growth rate, which showed that both enzyme induction and sugar transport were limiting. A set of point mutations in nagB that are known to affect the allosteric behavior of GlcN6P deaminase in vitro were transferred to the nagB gene on the Escherichia coli chromosome, and their effects on the growth rates were measured. Mutants in which the substrate-induced positive cooperativity of NagB was reduced or abolished grew even more slowly on GlcN than on GlcNAc or did not grow at all on GlcN. Increasing the amount of the deaminase by using a nagC or nagA mutation to derepress the nag operon improved growth. For some mutants, a nagA mutation, which caused the accumulation of the allosteric activator GlcNAc6P and permitted allosteric activation, had a stronger effect than nagC. The effects of the mutations on growth in vivo are discussed in light of their in vitro kinetics.

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Triphenilphosphonium Analogs of Chloramphenicol as Dual-Acting Antimicrobial and Antiproliferating Agents

10.20944/preprints202104.0026.v1 ◽

2021 ◽

Author(s):

Julia A. Pavlova ◽

Zimfira Z. Khairullina ◽

Andrey G. Tereshchenkov ◽

Pavel A. Nazarov ◽

Dmitrii A. Lukianov ◽

...

Keyword(s):

Inhibitory Effect ◽

Resistant Bacteria ◽

Inhibit Protein Synthesis ◽

Translation Process ◽

Gram Positive Bacteria ◽

Biochemical Assays ◽

Bacterial Ribosome ◽

Derivatives Of

In the current work, in continuation of our recent research [1] we synthesized and studied new chimeric compounds comprising the ribosome-targeting antibiotic chloramphenicol (CHL) and the membrane-penetrating cation triphenylphosphonium (TPP) connected by alkyl linkers of different lengths. Using various biochemical assays, we showed that these CAM-Cn-TPP compounds bind to the bacterial ribosome, inhibit protein synthesis in vitro and in vivo in a way similar to that of the parent CHL, and significantly decrease membrane potential. Similar to CAM-C4-TPP, the mode of action of CAM-C10-TPP and CAM-C14-TPP on bacterial ribosomes differ from that of CHL. By simulating the dynamics of complexes of CAM-Cn-TPP with bacterial ribosomes, we have proposed a possible explanation for the specificity of the action of these analogs on the translation process. CAM-C10-TPP and CAM-C14-TPP stronger inhibit the growth of the Gram-positive bacteria in comparison to the CHL and suppress some strains of CHL-resistant bacteria. Thus, we have shown that TPP derivatives of CHL are dual-acting compounds that target the ribosomes and the cellular membranes of bacteria. The TPP fragment of CAM-Cn-TPP compounds contributes to the inhibitory effect on bacteria. Moreover, since the mitochondria of eukaryotic cells have qualities similar to those of their prokaryotic ancestors, we demonstrate the possibility of targeting chemoresistant cancer cells with these compounds.

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