• Publication type
• Abstracts MeSH

The minor phospholipid, phosphatidylinositol 4-phosphate (PI4P), is emerging as a key regulator of lipid transfer in ER-membrane contact sites. Four different phosphatidylinositol 4-kinase (PI4K) enzymes generate PI4P in different membrane compartments supporting distinct cellular processes, many of which are crucial for the maintenance of cellular integrity but also hijacked by intracellular pathogens. While type III PI4Ks have been targeted by small molecular inhibitors, thus helping decipher their importance in cellular physiology, no inhibitors are available for the type II PI4Ks, which hinders investigations into their cellular functions. Here, we describe the identification of small molecular inhibitors of PI4K type II alpha (PI4K2A) by implementing a large scale small molecule high-throughput screening. A novel assay was developed that allows testing of selected inhibitors against PI4K2A in intact cells using a bioluminescence resonance energy transfer approach adapted to plate readers. The compounds disclosed here will pave the way to the optimization of PI4K2A inhibitors that can be used in cellular and animal studies to better understand the role of this enzyme in both normal and pathological states.

• MeSH
• 1-Phosphatidylinositol 4-Kinase antagonists & inhibitors   chemistry   metabolism   MeSH
• Biological Transport MeSH
• Chlorocebus aethiops MeSH
• COS Cells MeSH
• Endosomes drug effects   metabolism   MeSH
• Golgi Apparatus drug effects   metabolism   MeSH
• HEK293 Cells MeSH
• Enzyme Inhibitors metabolism   pharmacology   MeSH
• Protein Conformation MeSH
• Humans MeSH
• Drug Evaluation, Preclinical MeSH
• High-Throughput Screening Assays * MeSH
• Molecular Docking Simulation MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Intramural MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH

Electron microscopy in situ hybridization (EM-ISH) represents a powerful method that enables the localization of specific sequences of nucleic acids at high resolution. We provide here an overview of three different nonisotopic EM-ISH approaches that allow the visualization of nucleic acid sequences in cells. A comparison of various methods with respect to their sensitivity and the structural preservation of the sample is presented, with the aim of helping the reader to choose a convenient hybridization procedure. The post-embedding EM-ISH protocol that currently represents the most widely used technique is described in detail, with a special emphasis on the organization of the cell nucleus.

• MeSH
• Chlorocebus aethiops MeSH
• COS Cells MeSH
• Nucleic Acid Denaturation MeSH
• Deoxyribonuclease I MeSH
• DNA genetics   ultrastructure   MeSH
• Cryoelectron Microscopy methods   MeSH
• Microscopy, Electron methods   MeSH
• Endopeptidase K MeSH
• Financing, Organized MeSH
• Fixatives MeSH
• Genes, rRNA MeSH
• HeLa Cells MeSH
• HIV-1 genetics   MeSH
• In Situ Hybridization methods   MeSH
• Humans MeSH
• Microtomy methods   MeSH
• Molecular Probes MeSH
• Ribonuclease, Pancreatic MeSH
• RNA, Viral genetics   ultrastructure   MeSH
• RNA genetics   ultrastructure   MeSH
• Plastic Embedding MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Comparative Study MeSH

To biochemically and structurally characterize viral intracytoplasmic particles (ICAPs), a sample of high purity and homogeneity is usually required. Production of ICAPs in the system closely related to their natural host cells is crucial for the analysis of host-cell binding proteins involved in ICAPs assembly, transport and budding. However, this approach is often hampered by problems with low yield of the ICAPs due to either low expression or fast release from the host cell. Another obstacle may be a low stability or fragility of the intracellular particles. The published methods for ICAPs isolation often involved several time-consuming centrifugation steps yielding damaged particles. Other papers describe the ICAPs production in non-natural host cells. Here, we optimized the method for purification of unstable Mason-Pfizer monkey virus (M-PMV) ICAPs from non-human primate derived cells, commonly used to study MPMV replication i.e. African green monkey kidney fibroblast cell line (COS-1). Our simple and rapid procedure involved separation of the intracytoplasmic particles from the cell debris and organelles by differential, low-speed centrifugation, their purification using sucrose velocity gradient and final concentrating by low-speed centrifugation. Importantly, the method was established for unstable and fragile M-PMV intracytoplasmic particles. Therefore, it may be suitable for isolation of ICAPs of other viruses.

• MeSH
• Centrifugation methods   MeSH
• Chlorocebus aethiops MeSH
• COS Cells MeSH
• Cytoplasm virology   MeSH
• Humans MeSH
• Mason-Pfizer monkey virus isolation & purification   physiology   MeSH
• Virus Replication MeSH
• Virus Assembly MeSH
• Virology methods   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

• MeSH
• Biomechanical Phenomena MeSH
• Chlorocebus aethiops MeSH
• COS Cells MeSH
• Compressive Strength * MeSH
• Cell Survival MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

The coexistence of lipid domains with different degrees of lipid packing in the plasma membrane of mammalian cells has been postulated, but direct evidence has so far been challenging to obtain because of the small size and short lifetime of these domains in live cells. Here, we use fluorescence spectral correlation spectroscopy in conjunction with a probe sensitive to the membrane environment to quantify spectral fluctuations associated with dynamics of membrane domains in live cells. With this method, we show that membrane domains are present in live COS-7 cells and have a lifetime lower bound of 5.90 and 14.69 ms for the ordered and disordered phases, respectively. Comparisons to simulations indicate that the underlying mechanism of these fluctuations is complex but qualitatively described by a combination of dye diffusion between membrane domains as well as the motion of domains within the membrane.

• MeSH
• Benzoxazines chemistry   MeSH
• Cell Membrane chemistry   MeSH
• Chlorocebus aethiops MeSH
• COS Cells MeSH
• Spectrometry, Fluorescence MeSH
• Quaternary Ammonium Compounds chemistry   MeSH
• Membrane Lipids chemistry   MeSH
• Cell Survival MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

We have shown previously that a variant allele of the short-chain acyl-CoA dehydrogenase ( SCAD ) gene, 625G-->A, is present in homozygous form in 7% of control individuals and in 60% of 135 patients with elevated urinary excretion of ethylmalonic acid (EMA). We have now characterized three disease-causing mutations (confirmed by lack of enzyme activity after expression in COS-7 cells) and a new susceptibility variant in the SCAD gene of two patients with SCAD deficiency, and investigated their frequency in patients with elevated EMA excretion. The first SCAD-deficient patient was a compound heterozygote for two mutations, 274G-->T and 529T-->C. These mutations were not present in 98 normal control alleles, but the 529T-->C mutation was found in one allele among 133 patients with elevated EMA excretion. The second patient carried a 1147C-->T mutation and the 625G-->A polymorphism in one allele, and a single point mutation, 511C-->T, in the other. The 1147C-->T mutation was not present in 98 normal alleles, but was detected in three alleles of 133 patients with elevated EMA excretion, consistently as a 625A-1147T allele. On the other hand, the 511C-->T mutation was present in 13 of 130 and 15 of 67 625G alleles, respectively, of normal controls and patients with elevated EMA excretion, and was never associated with the 625A variant allele. This over-representation of the haplotype 511T-625G among the common 625G alleles in patients compared with controls was significant ( P < 0.02), suggesting that the allele 511T-625G-like 511C-625A-confers susceptibility to ethylmalonic aciduria. Expression of the variant R147W SCAD protein, encoded by the 511T-625G allele, in COS-7 cells showed 45% activity at 37 degrees C in comparison with the wild-type protein, comparable levels of activity at 26 degrees C, and 13% activity when incubated at 41 degrees C. This temperature profile is different from that observed for the variant G185S SCAD protein, encoded by the 511C-625A allele, where higher than normal activity was found at 26 and 37 degrees C, and 58% activity was present at 41 degrees C. These results corroborate the notion that the 511C-625A variant allele is one of the possible underlying causes of ethylmalonic aciduria, and suggest that the 511C-->T mutation represents a second susceptibility variation in the SCAD gene. We conclude that ethylmalonic aciduria, a commonly detected biochemical phenotype, is a complex multifactorial/polygenic condition where, in addition to the emerging role of SCAD susceptibility alleles, other genetic and environmental factors are involved.

• MeSH
• Acyl-CoA Dehydrogenase MeSH
• Acyl-CoA Dehydrogenases genetics   deficiency   MeSH
• Alleles MeSH
• COS Cells MeSH
• Fibroblasts metabolism   MeSH
• Gene Frequency MeSH
• Infant MeSH
• DNA, Complementary analysis   MeSH
• Cells, Cultured MeSH
• Humans MeSH
• Malonates * urine   MeSH
• Mutation MeSH
• Infant, Newborn MeSH
• Temperature MeSH
• Blotting, Western MeSH
• Animals MeSH
• Check Tag
• Infant MeSH
• Humans MeSH
• Male MeSH
• Infant, Newborn MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Case Reports MeSH
• Research Support, Non-U.S. Gov't MeSH

Lobeline is a plant alkaloid known to interact with cholinergic system. The effect of lobeline on neuronal α3β4 receptors expressed in COS cells and muscle embryonic αβγδ receptors naturally expressed in TE671 cells was studied using a patch-clamp technique. Our results show that lobeline inhibited responses to acetylcholine in human embryonic muscle nicotinic receptor in a pseudo-noncompetitive manner. The responses of rat neuronal α3β4 receptors to a low concentration of acetylcholine were potentiated by a mixed occupation mechanism that corresponds to "competitive potentiation". This potentiation turned into voltage-dependent inhibition for α3β4 receptors was activated by a high concentration of acetylcholine.

• MeSH
• Chlorocebus aethiops MeSH
• COS Cells MeSH
• Rats MeSH
• Humans MeSH
• Lobeline pharmacology   MeSH
• Neurons drug effects   metabolism   MeSH
• Receptors, Nicotinic metabolism   MeSH
• Nicotinic Agonists pharmacology   MeSH
• Organ Specificity MeSH
• Muscles drug effects   metabolism   MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

The effect of lobeline on rat α4β2 nicotinic receptors expressed in COS cells was studied using the patch-clamp technique. Currents were recorded in whole-cell mode 2-4 days after cell transfection by plasmids coding the α4β2 combination of receptor subunits. In cells sensitive to acetylcholine, the application of lobeline evoked minor responses (up to 2% of maximal acetylcholine response). When acetylcholine was applied to the background of an already running application of lobeline, acetylcholine responses were inhibited in a concentration- and time dependent manner. However, when lobeline was applied simultaneously with acetylcholine without any prepulse or during an already running application of acetylcholine, the acetylcholine responses were potentiated up to 300-600% of that of the control. The site of lobeline action overlaps with the cholinergic site, as was proven by the partially protective effect of (+)-tubocurarine. Thus, lobeline can apparently desensitize receptors when applied alone (inhibition) whereas its binding to a second agonist site with the first one already occupied by acetylcholine leads to channel opening (potentiation).

• MeSH
• Acetylcholine pharmacology   MeSH
• Chlorocebus aethiops MeSH
• COS Cells MeSH
• Rats MeSH
• Lobeline pharmacology   MeSH
• Neurons metabolism   MeSH
• Receptors, Nicotinic metabolism   MeSH
• Nicotinic Agonists pharmacology   MeSH
• Nicotinic Antagonists pharmacology   MeSH
• Drug Synergism MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

In various mammals, enzymatically active and inactive members of family 18 glycosyl hydrolases, containing chitinases, have been identified. In man, chitotriosidase is the functional chitinolytic enzyme, whilst the homologous human cartilage 39-kDa glycoprotein (HC gp-39) does not exhibit chitinase activity and its function is unknown. This study establishes that HC gp-39 is a chitin-specific lectin. It is experimentally demonstrated that a single amino acid substitution in the catalytic centre of the 39-kDa isoform of chitotriosidase, which generates a similar sequence to that in HC gp-39, results in a loss of hydrolytic activity and creates the capacity to bind to chitin. The possible implication of the finding for chitinolytic and chitin-binding proteins that are produced in high quantities by activated macrophages are discussed.

• MeSH
• Adipokines MeSH
• Chitin * metabolism   MeSH
• COS Cells MeSH
• Glycoproteins metabolism   secretion   MeSH
• Hexosaminidases genetics   metabolism   secretion   MeSH
• Cells, Cultured MeSH
• Lectins MeSH
• Humans MeSH
• Macrophages enzymology   secretion   MeSH
• Molecular Sequence Data MeSH
• Mutagenesis, Site-Directed MeSH
• Amino Acid Sequence MeSH
• Sequence Homology, Amino Acid MeSH
• Binding Sites MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Research Support, Non-U.S. Gov't MeSH