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At least 1,225 records · Page 68Linked to original sources
PubMed2003

Regulation of translation of the head protein of T4 bacteriophage by specific binding of EF-Tu to a leader sequence.

Recent evidence indicates that translation elongation factor Tu (EF-Tu) has a role in the cell in addition to its well established role in translation. The translation factor binds to a specific region called the Gol region close to the N terminus of the T4 bacteriophage major head protein as the head protein emerges from the ribosome. This binding was discovered because EF-Tu bound to Gol peptide is the specific substrate of the Lit protease that cleaves the EF-Tu between amino acid residues Gly59 and lle60, blocking phage development. These experiments raised the question of why the Gol region of the incipient head protein binds to EF-Tu, as binding to incipient proteins is not expected from the canonical role of EF-Tu. Here, we use gol-lacZ translational fusions to show that cleavage of EF-Tu in the complex with Gol peptide can block translation of a lacZ reporter gene fused translationally downstream of the Gol peptide that activated the cleavage. We propose a model to explain how binding of EF-Tu to the emerging Gol peptide could cause translation to pause temporarily and allow time for the leader polypeptide to bind to the GroEL chaperonin before translation continues, allowing cotranslation of the head protein with its insertion into the GroEL chaperonin chamber, and preventing premature synthesis and precipitation of the head protein. Cleavage of EF-Tu in the complex would block translation of the head protein and therefore development of the infecting phage. Experiments are presented that confirm two predictions of this model. Considering the evolutionary conservation of the components of this system, this novel regulatory mechanism could be used in other situations, both in bacteria and eukaryotes, where proteins are cotranslated with their insertion into cellular structures.

Bacterial Proteins↗
PubMed2006

Atomistic and coarse-grained analysis of double spectrin repeat units: the molecular origins of flexibility.

Spectrin is an ubiquitous protein in metazoan cells, and its flexibility is one of the keys to maintaining cellular structure and organization. Both alpha-spectrin and beta-spectrin polypeptides consist primarily of triple coiled-coil modular repeat units, and two important factors that determine spectrin flexibility are the bending flexibility between two consecutive repeat units and the conformational flexibility of individual repeat units. Atomistic molecular dynamics (MD) simulations are used here to study double spectrin repeat units (DSRUs) from the human erythrocyte beta-spectrin (HEbeta89) and the chicken brain alpha-spectrin (CBalpha1617). From the results of MD simulations, a highly conserved Trp residue in the A-helix of most repeat units that has been suggested to be important in conferring stability to the coiled-coil structures is found not to have a significant effect on the conformational flexibility of individual repeat units. Characterization of the bending flexibility for two consecutive repeats of spectrin via atomistic simulations and coarse-grained (CG) modeling indicate that the bending flexibility is governed by the interactions between the AB-loop of the first repeat unit, the BC-loop of the second repeat unit and the linker region. Specifically, interactions between residues in these regions can lead to a strong directionality in the bending behavior of two repeat units. The biological implications of these finding are discussed.

Amino Acid Sequence↗
PubMed2004

A new strategy for analysis of phenotype marker antigens in hollow neurospheres.

Changes in expression of phenotypic markers characterizing the cells comprising intact neurospheres are difficult to determine easily and accurately. The problem is compounded by the diversity of cell populations and phenotypes involved, and consequent non-uniform protein expression across the neurosphere wall, or around the circumference. Therefore, interpreting the effects of phenotype modifying conditions has been a complex and demanding task. Here, we report a novel direct method for measuring protein expression in immunofluoerescently labeled intact neurospheres by densitometric image analysis of optical cross-sections, obtained by scanning laser confocal microscopy. To demonstrate our methodology, hollow human neurospheres were exposed to basic fibroblast growth factor (FGF2), which reportedly induces neuronal commitment in monolayer cultures of neuroprogenitor cells derived from neurospheres. We determined that this treatment downregulated nestin and vimentin, protein markers accepted to indicate an immature, uncommitted phenotype. Neuron specific enolase was only marginally affected. Our strategy allows quantitation of changes in expression of marker proteins that is comparable to Western blot analysis. In addition to discriminating heterogeneity in protein expression, suitable optics may allow the resolution down to single cell level. We propose that this novel strategy, with or without confocal microscopy, may be applied to other biological systems. Analysis of protein expression by the cells comprising tubular or cylindrical cellular structures, or approximately spherical cell aggregates, can be performed efficiently using a small sample size.

Antigens↗
PubMed2026

Temporal proteomic analysis reveals a three-phase adaptation strategy in Phytophthora cinnamomi during salinity stress.

Phytophthora cinnamomi, a highly invasive hemibiotrophic oomycete, threatens global agriculture, forestry, and native ecosystems. Although drought and temperature effects on P. cinnamomi-host interactions are well studied, current knowledge of abiotic stress responses in P. cinnamomi remains largely centered on infection and phytopathology, with limited molecular insight into the pathogen's direct response to salinity independent of its host. To address this gap, we combined growth assays, time-resolved proteomics, and network analysis to define how P. cinnamomi responds and adapts to salinity exposure. Growth assays showed that NaCl-modified agar enhanced mycelial expansion in a concentration-dependent manner, with 100 mM NaCl significantly increasing growth at 48, 72, and 96 h compared with controls, while 50 mM NaCl remained comparable to control conditions. Temporal proteomic analysis of 100 mM NaCl treatment at 0, 1, 6, 12, and 24 h post treatment revealed dynamic shifts in protein abundance. Early induction of ROS (Reactive Oxygen Species)-detoxifying enzymes, including glutathione S-transferases and peroxidases, was consistent with ROS-specific staining assays. Network analysis identified modules enriched for redox regulation, ATP generation, ion transport, and translational control, highlighting multi-layered adaptation to elevated NaCl levels. Notably, clusters of conserved hypothetical proteins were strongly upregulated, indicating unexplored stress tolerance components in Phytophthora species. Here, we propose that P. cinnamomi rapidly activates a three-phase strategy involving metabolism readjustments, redox defenses, and cellular structure alterations under salinity conditions. With increasing soil salinization due to climate change, our study provides first mechanistic insights into P. cinnamomi's adaptive plasticity and ecological resilience to abiotic stress. SIGNIFICANCE: This study represents the first temporal proteomic analysis of salinity stress adaptation in Phytophthora cinnamomi, revealing a sophisticated three-phase adaptation strategy. This research fundamentally advances our understanding of how this globally destructive plant pathogen, P. cinnamomi, maintains environmental resilience. Our findings reveal proteome remodelling as a mechanistic framework for understanding stress tolerance in oomycetes, a group of microorganisms responsible for some of the world's most destructive agricultural and forest diseases. Our results show proteins involved in emergency damage control through metabolic recalibration to sustained adaptation. These findings have relevance for predicting pathogen behavior under climate change scenarios, where increasing soil salinity threatens agricultural productivity while simultaneously enhancing pathogen survival and virulence. Understanding how P. cinnamomi responds to prolonged salinity exposure may inform targeted biocontrol strategies and improve predictive models of disease pressure in salt-affected agricultural regions. The temporal analysis framework we present offers a broadly applicable approach for understanding microbial stress adaptation, with implications extending beyond plant pathology to environmental microbiology and biotechnology applications where stress tolerance is paramount.

Phytophthora↗
PubMed2005

A public software for energy filtering transmission electron tomography (EFTET-J): application to the study of granular inclusions in bacteria from Riftia pachyptila.

Energy-filtering transmission electron microscopy (EFTEM) allows the determination of elemental distributions out of a sequence of energy filtered images. Combined with electron tomography, EFTEM is a powerful tool to obtain three-dimensional chemical maps from sub-cellular structures. However, there is no existing software in the public-domain for the computation and analysis of 3D-chemical maps. Here, we present a Java-based program to compute 3D-elemental distribution. This program is available as a set of plug-ins for the public-domain Java image processing program Image J inspired by NIH Image. Its implemented algorithms have been successfully applied to the three-dimensional localization of iron granules in semi thin (200 nm) epon sections from the vent worm Riftia pachyptalia.

Algorithms↗
PubMed2006

Correlative microXRF and optical immunofluorescence microscopy of adherent cells labeled with ultrasmall gold particles.

Synchrotron-based X-ray fluorescence microscopy (microXRF) is a powerful tool to study the two-dimensional distribution of a wide range of biologically relevant elements in tissues and cells. By growing mouse fibroblast cells directly on formvar-carbon coated electron microscopy grids, microXRF elemental maps with well-defined subcellular resolution were obtained. In order to colocalize the elemental distribution with the location of specific cellular structures and organelles, we explored the application of a commercially available secondary antibody conjugated to FluoroNanogold, a dual-label that combines a regular organic fluorophore with a 1.4 nm Au-cluster as xenobiotic label for microXRF imaging. Adherent mouse fibroblast cells were grown on silicon nitride windows serving as biocompatible XRF support substrate, and labeled with FluoroNanogold in combination with primary antibodies specific for mitochondria or the Golgi apparatus, respectively. Raster scanning of the in-air dried cells with an incident X-ray energy of 11.95 keV, sufficient to ensure excitation of the Au Lalpha line, provided two-dimensional maps with submicron resolution for Au as well as for most biologically relevant elements. MicroXRF proved to be sufficiently sensitive to image the location and structural details of the Au-labeled organelles, which correlated well with the subcellular distribution visualized by means of optical fluorescence microscopy.

Animals↗
PubMed2006

Adaptive mechanisms to oxidative stress during aging.

Whether or not oxidative stress is the cause of the aging process, as proposed by the oxidative stress theory of aging remains unknown; but accumulated evidence overwhelmingly identifies increased oxidative stress with age as a source of damage to cellular structure and function. From an evolutionary perspective, the utilization of oxygen as a life supporting means makes oxidative stress an inescapable part of an organism's biological system. The inseparability of oxidative stress from the biological system can be viewed as an adaptive response that all aerobic organisms undergo to ward-off the potentially harmful effects of oxygen and its derivatives, including free radicals. The organism's adaptive mechanisms include an intricate network of defenses that regulate and guard against any over-acting oxidative reactions to ensure its survival. This review discusses and illustrates several adaptive responses at various levels (from gene regulation to physical exercise) that organisms use as part of their survival strategy.

Aging↗
PubMed2004

A molecular basis for embryo apposition at the luminal epithelium.

To obtain a gene expression profile during embryo apposition to the luminal epithelium, we isolated mouse luminal epithelium from implantation (IM) and interimplantation (INTER) sites using laser capture microdissection (LCM), and analyzed their gene expression by microarray analysis. IM and INTER sites were sampled on day 4.5 after mating of female mice with fertile males (day 0.5 = vaginal plug). RNA was extracted, amplified, labeled, and hybridized to microarrays and results were analyzed using the significance analysis of microarrays (SAM) method. Comparison of IM and INTER sites by SAM identified 73 genes most highly ranked at IM, while 13 genes most highly expressed at the INTER sites, within the estimated false discovery rate (FDR) of 0.163. Among 73 genes at IM, 20 were ESTs or were of unknown function, and the remain 53 genes had known functions mainly relating to cellular structuring and others such as cell cycling, gene/protein expression, immune responses, invasion, metabolism, oxidative stress, or signal transduction. Specifically, of the 24 structural genes, 14 were implicated in extracellular matrix and tissue remodeling. Meanwhile, of the 13 genes that were highly expressed at INTER, eight were ESTs or of unknown function, and the remaining five were implicated in metabolism, signal transduction, and gene/protein expression. Among these 58 (53 + 5) genes with known functions, 13 genes (22.4%) were associated with Ca2+ for their function. Results of the present study suggest that (1) at IM sites, active tissue remodeling is occurring for embryo invasion while the INTER sites are relatively quiescent and (2) Ca2+ may be a vital regulatory factor in the apposition process. Investigations of human homologues of those genes expressed in the mouse luminal epithelium during apposition may help to understand the implantation process and/or implantation failure in humans.

Animals↗
PubMed2004

Microarray expression analysis of gad mice implicates involvement of Parkinson's disease associated UCH-L1 in multiple metabolic pathways.

Parkinson's disease (PD) is thought to be caused by environmental and genetic factors. Mutations in four genes, alpha-synuclein, parkin, DJ-1, and UCH-L1, have been identified in autosomal inherited forms of PD. The pathogenetic cause for the loss of neuronal cells in PD patients, however, remains to be determined. Due to the rarity of mutations in humans with PD, the analysis of animal models might help to further gain insights into the pathogenesis of familial PD. For UCH-L1, deficiency has been described in gad mice leading to axonal degeneration and formation of spheroid bodies in nerve terminals. Here, we investigated the gene expression pattern of the brain of 3-month-old Uch-l1-deficient gracile axonal dystrophy (gad) mice by microarray analysis. A total of 146 genes were differentially regulated by at least a 1.4-fold change with 103 being up-regulated and 43 being down-regulated compared with age and sex matched wildtype littermate mice. The gene products with altered expression are involved in protein degradation, cell cycle, vesicle transport, cellular structure, signal transduction, and transcription regulation. Most of the genes were modestly regulated, which is in agreement that severe alteration of these pathways might be lethal. Among the genes most significantly down-regulated is the brain-derived neurotrophic factor which might be one aspect of the pathogenesis in gad mice. Interestingly, several subunits of the transcription factor CCAAT/enhancer binding protein are up-regulated, which plays a central role in most altered pathways.

Animals↗
PubMed2004

Presence of translation elongation factor-1A (eEF1A) in the excitatory postsynaptic density of rat cerebral cortex.

The postsynaptic density (PSD) is a proteinaceous cellular structure that is specialized for postsynaptic signal transduction. Here, we show that eukaryotic translation factor-1A (eEF1A; formerly known as eEF-1alpha) is associated with the excitatory PSD in rat forebrain. Immunoblot analysis showed that eEF1A in the PSD fraction is enriched over homogenate. Salt (1.0M NaCl), but not non-ionic detergents such as Triton X-100 (1.0%) and n-octyl glucoside (1.0%), could dissociate eEF1A from the PSD core. In cultured cortical neurons, eEF1A was colocalized with postsynaptic markers (PSD95 and SynGAPalpha), but not with a presynaptic marker (synaptophysin). These results indicate that eEF1A is present in the PSD of the excitatory synapses.

Animals↗
PubMed2006

Nasal polyp diseases in allergic and nonallergic patients and steroid therapy.

OBJECTIVE: To investigate widespread disease causes, cellular-structural differences, and steroid response of nasal polyps (NPs). METHOD: Study group consisted of NPs, allergic-NPs, NPs with steroid therapy (ST), antrochoanal polyp (ACP), and controls. We investigated stromal eosinophil, mast cell, CD4+ and CD8+ cell counts and presence of squamous metaplasia, Ki-67 expression, intraepithelial eosinophils-mast cells, epithelial damage, edema, fibrosis, hyalinization, polymorphonuclear leukocyte, and glandular hyperplasia. RESULTS: In allergic-NPs, intraepithelial eosinophils and epithelial damage CD4+ were significantly higher than NPs and also, eosinophils, mast cells, intraepithelial eosinophils, and epithelial damage were significantly higher than ACP. Only stromal eosinophilic infiltration was significantly higher in NPs than ACP. There was significant increased glandular hyperplasia and decreased intraepithelial eosinophils, mast cells, CD4+ cells, squamous metaplasia, and epithelial damage with ST in allergic-NPs. There were no significant differences with ST in NPs. CONCLUSION: NPs in allergic and nonallergic patients may differ in their histology and in their histologic responses to ST. EBM RATING: B-3b.

Adolescent↗
PubMed2005

Poly(ADP-ribose) polymerases: homology, structural domains and functions. Novel therapeutical applications.

Poly(ADP-ribose) polymerases (PARPs) are a family of enzymes, which show differences in structure, cellular location and functions. However, all these enzymes possess poly(ADP-ribosyl)ation activity. Overactivation of PARP enzymes has been implicated in the pathogenesis of several diseases, including stroke, myocardial infarction, diabetes, shock, neurodegenerative disorder and allergy. The best studied of these enzymes (PARP-1) is involved in the cellular response to DNA damage so that in the event of irreparable DNA damage overactivation of PARP-1 leads to necrotic cell death. Inhibitors of PARP-1 activity in combination with DNA-binding antitumor drugs may constitute a suitable strategy in cancer chemotherapy. In addition, PARP inhibitors may be also useful to restore cellular functions in several pathophysiological states and diseases. This review gives an update of the state-of-the-art concerning PARP enzymes and their exploitation as pharmacological targets in several illnesses.

Cell Death↗
PubMed2004

Role of oxidative stress in neurodegeneration: recent developments in assay methods for oxidative stress and nutraceutical antioxidants.

Reactive oxygen species (ROS) are produced in the course of normal metabolism and they serve important physiological functions. However, because of their high reactivity, accumulation of ROS beyond the immediate needs of the cell may affect cellular structure and functional integrity, by bringing about oxidative degradation of critical molecules, such as the DNA, proteins, and lipids. Although cells possess an intricate network of defense mechanisms to neutralize excess ROS and reduce oxidative stress, some tissues, especially the brain, are much more vulnerable to oxidative stress because of their elevated consumption of oxygen and the consequent generation of large amounts of ROS. For the same reason, the mitochondrial DNA (mtDNA) of brain cells is highly susceptible to structural alterations resulting in mitochondrial dysfunction. Several lines of evidence strongly suggest that these effects of ROS may be etiologically related to a number of neurodegenerative disorders. Nutraceutical antioxidants are dietary supplements that can exert positive pharmacological effects on specific human diseases by neutralizing the negative effects of ROS. The present communication concentrates on a review of recent concepts and methodological developments, some of them based on the results of work from our own laboratory, on the following aspects: (1) the complex interactions and complementary interrelationships between oxidative stress, mitochondrial dysfunction, and various forms of neural degeneration; (2) fractionation and isolation of substances with antioxidant properties from plant materials, which are extensively used in the human diet and, therefore, can be expected to be less toxic in any pharmacological intervention; (3) recent developments in methodologies that can be used for the assay of oxidative stress and determination of biological activities of exogenous and endogenous antioxidants; and (4) presentation of simple procedures based on polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) of the resulting amplicon for investigations of structural alterations in mtDNA.

Animals↗
PubMed2005

The presence of acetylated tubulin in the pig thymus.

Immunohistochemical localisation of acetylated alpha-tubulin was investigated in pig thymus using a mouse monoclonal antibody. Positive reaction to acetylated tubulin was observed in the cells involved in Hassall's bodies localized in the medulla region. Reacting product displayed a homogenous, fine or coarse granular character. The reacting peripheral cells of Hassall's bodies produced crescent or annular formations. Inside the Hassall's bodies the centrally located cells were unreactive. The presence of the acetylated tubulin confirms positive cellular structures in these cells. Differences in the character and intensity of staining in the epithelial cells may confirm transformation of the epithelial cells during the formation of the Hassall's bodies and/or the presence of the neural crest derivative in the development of the thymus.

Acetylation↗
PubMed2006

Structure of artemin complexed with its receptor GFRalpha3: convergent recognition of glial cell line-derived neurotrophic factors.

Artemin (ARTN) is a member of the glial cell line-derived neurotrophic factor (GDNF) family ligands (GFLs) which regulate the development and maintenance of many neuronal populations in the mammalian nervous system. Here we report the 1.92 A crystal structure of the complex formed between ARTN and its receptor GFRalpha3, which is the initiating step in the formation of a ternary signaling complex containing the shared RET receptor. It represents a new receptor-ligand interaction mode for the TGF-beta superfamily that reveals both conserved and specificity-determining anchor points for all GFL-GFRalpha pairs. In tandem with the complex structure, cellular studies using receptor chimeras implicate dyad-symmetric composite interfaces for recruitment and dimerization of RET, leading to intracellular signaling. These studies should facilitate the functional dissection of the specific versus pleiotropic roles of this system in neurobiology, as well as its exploitation for therapeutic applications.

Amino Acid Sequence↗
PubMed2003

Quantitative cell biology with the Virtual Cell.

Cell biological processes are controlled by an interacting set of biochemical and electrophysiological events that are distributed within complex cellular structures. Computational models, comprising quantitative data on the interacting molecular participants in these events, provide a means for applying the scientific method to these complex systems. The Virtual Cell is a computational environment designed for cell biologists, to facilitate the construction of models and the generation of predictive simulations from them. This review summarizes how a Virtual Cell model is assembled and describes the physical principles underlying the calculations that are performed. Applications to problems in nucleocytoplasmic transport and intracellular calcium dynamics will illustrate the power of this paradigm for elucidating cell biology.

Active Transport, Cell Nucleus↗
PubMed2004

The glamour and gloom of glycogen synthase kinase-3.

Glycogen synthase kinase-3 (GSK3) is now recognized as a key component of a surprisingly large number of cellular processes and diseases. Several mechanisms play a part in controlling the actions of GSK3, including phosphorylation, protein complex formation, and subcellular distribution. These are used to control and direct the far-reaching influences of GSK3 on cellular structure, growth, motility and apoptosis. Dysregulation of GSK3 is linked to several prevalent pathological conditions, such as diabetes and/or insulin resistance, and Alzheimer's disease. Therefore, much effort is currently directed towards understanding the functions and control of GSK3, and identifying methods capable of diminishing the deleterious impact of GSK3 in pathological conditions.

Alzheimer Disease↗