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At least 37 records · Page 2Linked to original sources
PubMed1975

3-Deoxy-D-arabino-heptulosonic acid 7-phosphate synthase mutants of Salmonella typhimurium.

The first committed step of aromatic amino acid biosynthesis in Salmonella typhimurium was shown to be catalyzed by three isoenzymes of 3-deoxy-D-arabino-heptulosonic acid 7-phosphate (DAHP) synthase. Mutations in each of the genes specifying the isoenzymes were isolated and mapped. aroG, the structural gene for the phenylalanine-inhibitable isoenzyme, was linked to gal, and aroH, the structural gene for the tryptophan-inhibitable isoenzyme, was linked to aroE. aroF, the structural gene for the tyrosine-inhibitable isoenzyme, was linked to pheA and tyrA, which specify the phenylalanine- and tyrosine-specific branch-point enzymes, respectively. The phenylalanine-inhibitable isoenzyme was the predominant DAHP synthase in wild-type cells, and only the tryosine-inhibitable isoenzyme was completely repressed, as well as inhibited, by low levels of its allosteric effector. The DAHP synthase isoenzymes were separated by chromatography on diethylaminoethyl-cellulose with a phosphate gradient which contained enolpyruvate phosphate to protect the otherwise unstable phenylalanine-inhibitable isoenzyme. No cross-inhibition of either the tyrosine- or phenylalanine-inhibitable isoenzyme was observed at inhibitor concentrations up to 1 mM. The tryptophan-inhibitable isoenzyme was partially purified from extracts of a strain lacking the other two isoenzymes and shown to be inhibited about 30% by 1 mM tryptophan. A preliminary study of interference by tryptophan in the periodate-thiobarbiturate assay for DAHP suggested a combined effect of tryptophan and erythrose 4-phosphate, or an aldehydic compound resulting from degradation of erythrose 4-phosphate by periodate.

3-Deoxy-7-Phosphoheptulonate Synthase↗
PubMed1975

Regulation of dihydrodipicolinate synthase during growth and sporulation of Bacillus cereus.

A four- to sixfold increase in specific activity of dihydrodipicolinic acid synthase was observed during sporulation of Bacillus cereus. The enzyme from cells harvested before and after the increase in specific activity appeared to be very similar as judged by pH optima, heat denaturation kinetics, apparent Michaelis constants, chromatography on diethylaminoethyl-cellulose and Sephadex G-200, and polyacrylamide gel electrophoresis. Studies with various combinations of amino acids and one of the enzyme substrates, pyruvate, failed to give evidence for control of the enzyme by activation, inhibition, repression, induction, or stabilization. Omission of calcium from the sporulation medium had no significant effect on the specific activity pattern of the enzyme as a function of age of culture.

Amino Acids↗
PubMed1975

Purification, new assay, and properties of coenzyme A transferase from Peptostreptococcus elsdenii.

Coenzyme A (CoA) transferase from Peptostreptococcus elsdenii has been purified and crystallized, and some of its properties have been established. The work was facilitated by a newly developed coupled and continuous spectrophotometric assay in which the disappearance of added acrylate could be followed at 245 nm. The rate-limiting conversion of acetyl- and beta-hydroxypropionyl CoA to acrylyl CoA by CoA transferase was followed by the non-rate-limiting conversion to beta-hydroxypropionyl CoA by excess crotonase. Thus, a small priming quantity of acetyl CoA served to generate acrylyl CoA, which, by hydration, generated beta-hydroxypropionyl CoA. This product then served to generate more acrylyl CoA in cyclic fashion. The net result was the CoA transferase-limited conversion of acrylate to beta-hydroxypropionate. The purified transferase has a molecular weight of 125,000 and is composed of two subunits of 63,000 each, as determined by disc gel electrophoresis. Short-chain-length monocarboxylic acids are substrates, whereas dicarboxylic or beta-ketocarboxylic acids are not. The reaction kinetics are typical of a ping-pong bi bi mechanism composed of two half reactions linked by a covalent enzyme intermediate. Incubation of the transferase with acetyl CoA in the absence of a fatty acid acceptor yielded a stable intermediate which, by absorption spectrophotometry, radioactivity measurements, reduction with borohydride, reactivity with hydroxylamine, and catalytic activity, was identified as an enzyme-CoA compound. Kinetic constants for CoA transferase are: final specific activity, 110 U/mg of protein corresponding to 1.38 X 10(4) mumol of acrylate activated per mumol of transferase; Km for acrylate, 1.2 X 10(-3) M; Km for acetyl CoA (beta-hydroxypropionyl CoA), 2.4 X 10(-5) M.

Acetyl Coenzyme A↗
PubMed1975

Isolation and purification of Flavobacterium alpha-1,3-glucanase-hydrolyzing, insoluble, sticky glucan of Streptococcus mutans.

Studies were made on the physical and chemical properties of polysaccharides synthesized by cell-free extracts of Streptococcus mutans, Streptococcus sanguis, and Streptococcus sp. and their susceptibilities to dextranases. Among the polysaccharides examined, insoluble glucans were rather resistant to available dextranase preparations, and the insoluble, sticky glucan produced by S. mutans OMZ 176, which could be important in formation of dental plaques, was the most resistant. By enrichment culture of soil specimens, using OMZ 176 glucans as the sole carbon source, an organism was isolated that produced colonies surrounded by a clear lytic zone on opaque agar plates containing the OMZ 176 glucan. The organism was identified as a strain of Flavobacterium and named the Ek-14 bacterium. EK-14 bacterium was grown in Trypticase soy broth, and an enzyme capable of hydrolyzing the OMZ 176 glucan was concentrated from the culture supernatant and purified by negative adsorption on a diethylaminoethyl-cellulose (DE-32) column and gradient elution chromatography with a carboxymethyl-cellulose (CM-32) column. The enzyme was a basic protein with an isoelectric point of pH 8.5 and molecular weight of 65,000. Its optimum pH was 6.3 and its optimal temperature was 42 C. The purified enzyme released 11% of the total glucose residues of the OMZ 176 glucan as reducing sugars and solubilized about half of the substrate glucan. The products were found to be isomaltose, nigerose, and nigerotriose, with some oligosaccharides. The purified enzyme split the alpha-1,3-glucan endolytically and was inactive toward glucans containing alpha-1,6, alpha-1,4, beta-1,3, beta-1,4, and/or beta-1,6 bonds as the main linkages.

Cell-Free System↗
PubMed1975

Production, purification, and characterization of an extracellular chitosanase from Streptomyces.

The synthesis by Streptomyces sp. no. 6 of an extracellular chitosanase was induced by glucosamine. The enzyme was purified to homogeneity by Sephadex G-100, carboxymethyl-cellulose, and diethylaminoethyl-cellulose chromatography. The purified enzyme hydrolyzed chitosan (the beta-1,4-linked polymer of glucosamine) but not chitin nor carboxymethyl-cellulose. The only products of the hydrolysis detectable by paper chromatography were di- and triglucosamine. Sephadex G-100 chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the molecular weight of the enzyme was between 29,000 and 26,000. Acid hydrolysates of the enzyme contained no cysteic acid or glucosamine or other carbohydrate. At 25 C, maximum activity was obtained between pH 4.5 and 6.5. The enzymatic hydrolysis of chitosan occurred over a wide range of temperatures and was maximal at 60 C. The rate of the reaction was inhibited by concentrations of soluble chitosan higher than 0.5 g/liter. The apparent Km calculated from a Lineweaver-Burke plot was 0.688 g/liter at pH 5.5. The enzyme prevented spore germination and caused a significant decrease in the turbidity of germinated spore suspensions of the Mucor strains tested. Such a decrease was the result of a partial lysis of the cell wall.

Amino Sugars↗
PubMed1976

Physiological study of ergot: induction of alkaloid synthesis by tryptophan at the enzymatic level.

The enhancement of ergot alkaloid production by tryptophan and its analogues in both normal and high-phosphate cultures is more directly related to increased dimethylallyltryptophan (DMAT) synthetase activity rather than to a lack of regulation of the tryptophan biosynthetic enzymes. Thiotryptophan [beta-(1-benzo-thien-3-yl)-alanine] is rather ineffective in the end product regulation of tryptophan biosynthesis, whereas tryptophan and 5-methyltryptophan are potent effectors. The presence of increased levels of DMAT synthetase in ergot cultures supplemented with tryptophan or thiotryptophan, and to a lesser extent with 5-methyltryptophan, suggests that the induction effect involves de novo synthesis of the enzyme. Thiotryptophan and tryptophan but not 5-methyltryptophan can overcome the block of alkaloid synthesis by inorganic phosphate. The results with thiotryptophan indicate that the phosphate effect cannot be explained merely on the basis of a block of tryptophan synthesis.

3-Deoxy-7-Phosphoheptulonate Synthase↗
PubMed1975

Uukuniemi virus contains an RNA polymerase.

An RNA-dependent RNA polymerase activity has been found associated with Uukuniemi virions. The enzyme activity is expressed only after disrupting the virions with the nonionic detergent Triton X-100 and is absolutely dependent on Mn2+, whereas Mg2+ is not required, a finding that distinguishes this polymerase from those of other enveloped minus-strand RNA viruses. Within the range pH 7.2 to 8.5 no distinct optimum was found. The optimum temperature was between 37 and 40 C. The reaction was not inhibited by actinomycin D, rifampin, or DNase, whereas RNase was completely inhibitory. The partially RNase-resistant product consisted of rather small-sized RNA, which contained sequences complementary to Uukuniemi virus RNA as shown by hybridization to the template L, M, and S RNA species of Uukuniemi virus.

Arboviruses↗
PubMed1975

Nitrofurazone-reducing enzymes in E. coli and their role in drug activation in vivo.

Earlier work showed that Escherichia coli contains at least two enzymes which reduce nitrofurazone and other nitrofuran derivatives. One of these enzymes is lacking in some nitrofurazone-resistant mutant strains. We now report that there are three separable nitrofuran reductases in this organism: reductase I (mol. wt. approximately 50 000, insensitive to O2), reductase IIa (mol. wt. approximately 120 000, inhibited by oxygen), reductase IIb (mol. wt. approximately 700 000, inhibited by O2). Unstable metabolites formed during the reduction of nitrofurazone by preparations containing reductases IIa and IIb produce breaks in DNA in vitro. In vivo experiments with nitrofurazone-resistant strains, which lack reductase II but contain reductases IIa and IIb, demonstrated that lethality, mutation, and DNA breakage are all greatly increased when cultures are incubated under anaerobic conditions, i.e., conditions such that reductase II is active. These results provide further evidence for the importance of reductive activation of nitrofurazone.

Aerobiosis↗
PubMed1975

Enzymatic hydrolysis of agar: purification and characterization of neoagarobiose hydrolase and p-nitrophenyl alpha-galactoside hydrolase.

The mixture of polysaccharides in the gelling component of agar (agarose) is hydrolyzed to D-galactose and 3,6-anhydro-L-galactose by a series of hydrolytic enzymes obtained from Pseudomonas atlantica. The final degradative step in the pathway of agarose decomposition is the hydrolysis of the alpha-linkage in the dissaccharide neoagarobiose yielding D-galactose and 3,6-anhydro-L-galactose. Pseudomonas atlantica when grown on agar produces two specific enzymes, p-nitrophenyl alpha-galactose hydrolase and neoagarobiose hydrolase. The purification and partial characterization of both enzymes are presented.

Agar↗
PubMed1975

Regulation and properties of an invertase from Clostridium pasteurianum.

An intracellular invertase was induced in cultures of Clostridium pasteurianum utilizing sucrose as its carbon source for growth. This enzyme synthesis could be repressed by the addition of fructose of a sucrose-growing culture. In contrast, invertase activity was not affected by the addition of glucose to sucrose-growing cells and this enzyme could be induced in a glucose-metabolizing culture by the addition of sucrose. This enzyme was purified 10.5-fold over the induced lese, EC 3.2.1.26) by substrate-specificity studies. Invertase had a pH optimum of 6.5 and an apparent Km of 79.5 mM for sucrose, and required high concentration of potassium phosphate for maximum activity. Invertase was completely inactivated by a 2-min heat treatment at 60 degrees C. This enzyme was strongly inhibited by p-hydroxymercuribenzoate (pCMB) and weakly inhibited by 5,5'-dithiobis(2-nitrobenzoic acid), while cysteine could substantially reverse pCMB) inhibition, suggesting that sulfhydryl group(s) were necessary for invertase activity.

Cell-Free System↗
PubMed2003

Complexes between the LKB1 tumor suppressor, STRAD alpha/beta and MO25 alpha/beta are upstream kinases in the AMP-activated protein kinase cascade.

BACKGROUND: The AMP-activated protein kinase (AMPK) cascade is a sensor of cellular energy charge that acts as a 'metabolic master switch' and inhibits cell proliferation. Activation requires phosphorylation of Thr172 of AMPK within the activation loop by upstream kinases (AMPKKs) that have not been identified. Recently, we identified three related protein kinases acting upstream of the yeast homolog of AMPK. Although they do not have obvious mammalian homologs, they are related to LKB1, a tumor suppressor that is mutated in the human Peutz-Jeghers cancer syndrome. We recently showed that LKB1 exists as a complex with two accessory subunits, STRAD alpha/beta and MO25 alpha/beta. RESULTS: We report the following observations. First, two AMPKK activities purified from rat liver contain LKB1, STRAD alpha and MO25 alpha, and can be immunoprecipitated using anti-LKB1 antibodies. Second, both endogenous and recombinant complexes of LKB1, STRAD alpha/beta and MO25 alpha/beta activate AMPK via phosphorylation of Thr172. Third, catalytically active LKB1, STRAD alpha or STRAD beta and MO25 alpha or MO25 beta are required for full activity. Fourth, the AMPK-activating drugs AICA riboside and phenformin do not activate AMPK in HeLa cells (which lack LKB1), but activation can be restored by stably expressing wild-type, but not catalytically inactive, LKB1. Fifth, AICA riboside and phenformin fail to activate AMPK in immortalized fibroblasts from LKB1-knockout mouse embryos. CONCLUSIONS: These results provide the first description of a physiological substrate for the LKB1 tumor suppressor and suggest that it functions as an upstream regulator of AMPK. Our findings indicate that the tumors in Peutz-Jeghers syndrome could result from deficient activation of AMPK as a consequence of LKB1 inactivation.

AMP-Activated Protein Kinase Kinases↗
PubMed1976

On the nature of the presumed receptor for IgE on mast cells. III. Kinetics of the blocking of the PCA reaction by cell-free particulate preparations from rat peritoneal mast cells and effect of pH and calcium concentration on the reaction.

The 'binding' of IgE to particulate preparations derived from sonicated purified rat mast cells was measured by the blocking of PCA titrations of the supernatant solutions from incubations with such preparations. It was found that the PCA blocking reaction was inhibited by the addition of calcium ion to the incubations. The blocking reaction was strongly dependent on the pH of the incubations, being maximal at pH values lower than 5-0. The blocking reaction proceeded in a linear manner for at least 3 h provided that no more than 70 percent of the amount of IgE initially supplied had been removed by the particulate fraction. Only mast cell-derived preparations were capable of effecting PCA blocking.

Animals↗
PubMed1976

Inhibition of Newcastle disease virus replication by 6-azauridine. I. Inefficacy of purified uridine kinase, effect of adenosine-5'-triphosphate.

As distinct from cell-free extracts prepared from tumour cells, partially purified uridine kinase prepared from the same cells was not effective in 6-azauridine (AzUrd) inhibition of Newcastle disease virus (NDV) replication. This showed that uridine kinase was not the effective component of cell-free extracts. Adenosine-5'-triphosphate (ATP) was found to exert a synergic effect in combination with AzUrd in the inhibition of NDV replication.

Adenosine Triphosphate↗
PubMed2026

A liquid handling platform for standardized quantification of cell-free enzymatic activity encoded by antimicrobial resistance genes.

Antimicrobial resistance (AMR) is a growing global threat to human health, and rapid methods for characterizing emerging antimicrobial resistance genes (ARGs) are needed. Here, we develop a semi-automated workflow using cell-free gene expression systems to measure the activity of two ARGs encoded on plasmid DNA that produce rifampicin-inactivating and gentamicin-inactivating enzymes. We validated the use of a small benchtop Myra liquid handling system compared to manual pipetting, with no statistical differences observed. After optimizing the pre-incubation time of ARGs and dispensing protocol, expression of aac(3)-IIa increased the half-maximal inhibition concentration (IC50) of gentamicin by over 150-fold, whilst arr-3 increased the IC50 of rifampicin by ~20-fold compared to controls. This methodology for rapid, semi-automated ARG characterization offers a strategy to combat AMR by assessing novel ARGs identified through genomic surveillance or profiling activity of new or derivative antibiotics.

aminoglycoside resistance↗
PubMed2025

Detecting known neoepitopes, gene fusions, transposable elements, and circular RNAs in cell-free RNA.

MOTIVATION: Cancer is the second leading cause of death worldwide, and although there have been advances in treatments, including immunotherapies, these often require biopsies which can be costly and invasive to obtain. Due to lack of pre-emptive cancer detection methods, many cases of cancer are detected at a late stage when the definitive symptoms appear. Plasma samples are relatively easy to obtain, and they can be used to monitor the molecular signatures of ongoing processes in the body. Profiling cell-free DNA is a popular method for monitoring cancer, but only a few studies have explored the use of cell-free RNA (cfRNA), which shows the recent footprint of systemic transcription. RESULTS: Here, we developed FastNeo, a computational method for detecting known neoepitopes in human cfRNA. We show that neoepitopes and other biomarkers detected in cfRNA can discern Hepatocellular carcinoma patients from the healthy patients with a sensitivity of 0.84 and a specificity of 0.79. For colorectal cancer we achieve a sensitivity of 0.87 and a specificity of 0.8. An important advantage of our cfRNA based approach is that it also reports putative neoepitopes which are important for therapeutic purposes. AVAILABILITY AND IMPLEMENTATION: The FastNeo package is available at https://github.com/yashumayank/FastNeo and https://zenodo.org/records/11521368. The benchmark pipelines to detect Immune Epitope database and Tumor-Specific Neoantigen database neoepitopes using HaplotypeCaller, bcftools, and Lofreq, and to run FastNeo with STAR instead of Bowtie2 are also available in the above github repository.

Humans↗
PubMed2026

Aging and Reproductive Cancers: An Integrative View on Cell-Free DNA and Transposable Elements.

Aging is one of the strongest risk factors for cancer, and its impact is particularly evident in malignancies of the reproductive system. Ovarian, endometrial, cervical, vulvar, prostate, and penile cancers are mainly diagnosed in older adults and often show different clinical and biological features compared with the same tumors in younger patients. Aging is associated with hormonal changes, immune decline, epigenetic alterations, and accumulation of DNA damage, all of which contribute to cancer development and progression. At the same time, many older patients have frailty and multiple comorbidities, which can limit the use of screening programs and invasive diagnostic procedures. This often leads to delayed diagnosis and worse outcomes. Cell-free DNA (cfDNA) is a minimally invasive biomarker that can be obtained from blood samples and provides molecular information on both tumor and host tissues. Circulating DNA reflects tumor-specific alterations but is also influenced by aging-related changes in DNA release, fragmentation, and methylation. For this reason, aging must be considered when cfDNA-based biomarkers are applied in clinical practice. In this review, we describe how aging influences the biology of reproductive system cancers and how these processes are mirrored in cfDNA profiles. We focus on the clinical use of cfDNA for cancer detection and monitoring in older and fragile patients. Special attention is given to repetitive elements in cfDNA, which are strongly affected by aging and tumor-related epigenetic changes and can be detected with high sensitivity even when the tumor fraction is low. We propose an integrative mechanistic framework in which age-related epigenetic and genomic changes influence both tumor biology and cfDNA composition, with transposable elements acting as a central link between aging and cancer.

Humans↗
PubMed2024

Central conducting lymphatic anomaly: from bench to bedside.

Central conducting lymphatic anomaly (CCLA) is a complex lymphatic anomaly characterized by abnormalities of the central lymphatics and may present with nonimmune fetal hydrops, chylothorax, chylous ascites, or lymphedema. CCLA has historically been difficult to diagnose and treat; however, recent advances in imaging, such as dynamic contrast magnetic resonance lymphangiography, and in genomics, such as deep sequencing and utilization of cell-free DNA, have improved diagnosis and refined both genotype and phenotype. Furthermore, in vitro and in vivo models have confirmed genetic causes of CCLA, defined the underlying pathogenesis, and facilitated personalized medicine to improve outcomes. Basic, translational, and clinical science are essential for a bedside-to-bench and back approach for CCLA.

Cell-Free Nucleic Acids↗
PubMed2026

iMSC-derived extracellular vesicles and their miRNA cargo influence inflammation and oxidative damage in an in vitro osteoarthritis model.

Osteoarthritis is a multifactorial chronic joint disease characterized by progressive cartilage degradation and inflammation. Since there is no effective cure, emerging therapeutic approaches, such as mesenchymal stromal cells (MSCs) transplantation, are currently under investigation. However, the clinical translation of MSC-based therapies is hampered by several limitations, such as donor-dependent variability and heterogeneity related to tissue sources. To address these issues, MSCs derived from induced pluripotent stem cells (iMSCs) have been proposed as a more standardized and scalable alternative. Due to the risks of cell-based therapy, extracellular vesicles (EVs), particularly iMSC-EVs (iEVs), could represent a promising cell-free approach for OA treatment. The present study aimed at characterizing iMSC-derived EVs and evaluating their functional role in modulating inflammatory responses and redox balance in an in vitro OA model. Notably, recent evidence highlights the central role of EV-encapsulated microRNAs (EV-miRNAs) in mediating these effects. EVs isolated from iMSC conditioned media were characterized, and their miRNA content was analyzed at different culture passages. Selected miRNAs were subsequently assessed for their biological activity in an in vitro OA model, with a focus on their impact on inflammatory mediators and oxidative stress parameters. Specifically, six miRNAs such as hsa-miR-17-5p, hsa-miR-20a-5p, hsa-miR-21-5p, hsa-miR-29a-3p, hsa-miR-29b-3p, and hsa-miR-29c-3p differentially reflect the anti-inflammatory and antioxidant effects of iMSCs-EVs treatment, suggesting possible synergistic effects. Their combined effect in the in vitro model confirmed their potential modulation in the expression of pro-inflammatory cytokines. Furthermore, their treatment markedly reduced ROS accumulation and oxidative damage, while restoring antioxidant defense systems. These findings support the therapeutic potential of iMSC-derived EVs as a cell-free strategy for OA treatment. The miRNA cargo encapsulated within iEVs appears to play a pivotal role in modulating inflammation and oxidative stress, emphasizing their promise as a novel, minimally invasive approach for disease modification in OA.

MicroRNAs↗