Assembly of the Gag polyprotein into new viral particles in infected cells is a crucial step in the retroviral replication cycle. Currently, little is known about the onset of assembly in the cytosol. In this paper, we analyzed the cytosolic HIV-1 Gag fraction in real time in live cells using advanced fluctuation imaging methods and thereby provide detailed insights into the complex relationship between cytosolic Gag mobility, stoichiometry, and interactions.
View Article and Find Full Text PDFThe cellular endosomal sorting complex required for transport (ESCRT) machinery is involved in membrane budding processes, such as multivesicular biogenesis and cytokinesis. In HIV-infected cells, HIV-1 hijacks the ESCRT machinery to drive HIV release. Early in the HIV-1 assembly process, the ESCRT-I protein Tsg101 and the ESCRT-related protein ALIX are recruited to the assembly site.
View Article and Find Full Text PDFHIV (human immunodeficiency virus) diverts the cellular ESCRT (endosomal sorting complex required for transport) machinery to promote virion release from infected cells. The ESCRT consists of four heteromeric complexes (ESCRT-0 to ESCRT-III), which mediate different membrane abscission processes, most importantly formation of intralumenal vesicles at multivesicular bodies. The ATPase VPS4 (vacuolar protein sorting 4) acts at a late stage of ESCRT function, providing energy for ESCRT dissociation.
View Article and Find Full Text PDFAssembly and release of human immunodeficiency virus (HIV) occur at the plasma membrane of infected cells and are driven by the Gag polyprotein. Previous studies analyzed viral morphogenesis using biochemical methods and static images, while dynamic and kinetic information has been lacking until very recently. Using a combination of wide-field and total internal reflection fluorescence microscopy, we have investigated the assembly and release of fluorescently labeled HIV-1 at the plasma membrane of living cells with high time resolution.
View Article and Find Full Text PDFA colorful variety of fluorescent proteins (FPs) from marine invertebrates are utilized as genetically encoded markers for live cell imaging. The increased demand for advanced imaging techniques drives a continuous search for FPs with new and improved properties. Many useful FPs have been isolated from species adapted to sun-flooded habitats such as tropical coral reefs.
View Article and Find Full Text PDFFluorescent proteins (FPs) emitting in the far-red region of the spectrum are highly advantageous for whole-body imaging applications because scattering and absorption of long-wavelength light is markedly reduced in tissue. We characterized variants of the red fluorescent protein eqFP611 with bright fluorescence emission shifted up to 639 nm. The additional red shift is caused by a trans-cis isomerization of the chromophore.
View Article and Find Full Text PDFRecent years have witnessed enormous advances in fluorescence microscopy instrumentation and fluorescent marker development. 4Pi confocal microscopy with two-photon excitation features excellent optical sectioning in the axial direction, with a resolution in the 100 nm range. Here we apply this technique to cellular imaging with EosFP, a photoactivatable autofluorescent protein whose fluorescence emission wavelength can be switched from green (516 nm) to red (581 nm) by irradiation with 400-nm light.
View Article and Find Full Text PDFFor a variety of coral species, we have studied the molecular origin of their coloration to assess the contributions of host and symbiont pigments. For the corals Catalaphyllia jardinei and an orange-emitting color morph of Lobophyllia hemprichii, the pigments belong to a particular class of green fluorescent protein-like proteins that change their color from green to red upon irradiation with approximately 400 nm light. The optical absorption and emission properties of these proteins were characterized in detail.
View Article and Find Full Text PDFEosFP is a fluorescent protein from the coral Lobophyllia hemprichii that changes its fluorescence emission from green to red upon irradiation with near-UV light. Here we present the spectroscopic properties of wild-type EosFP and a variety of monomeric and dimeric mutants and provide a structural interpretation of its oligomerization and photoconversion, which is based on X-ray structure analysis of the green and red species that we reported recently. Because functional expression of the monomeric EosFP variant is limited to temperatures of 30 degrees C, we have developed a tandem dimer.
View Article and Find Full Text PDFEosFP is a novel fluorescent protein from the stony coral Lobophyllia hemprichii. Its gene was cloned in Escherichia coli to express the tetrameric wild-type protein. The protein emits strong green fluorescence (516 nm) that shifts toward red (581 nm) upon near-ultraviolet irradiation at ∼390 nm due to a photo-induced modification that involves a break in the peptide backbone next to the chromophore.
View Article and Find Full Text PDFThe red fluorescent protein (FP) eqFP611 from the sea anemone Entacmaea quadricolor shows favorable properties for applications as a molecular marker. Like other anthozoan FPs, it forms tetramers at physiological concentrations. The interactions among the monomers, however, are comparatively weak, as inferred from the dissociation into monomers in the presence of sodium dodecyl sulfate (SDS) or at high dilution.
View Article and Find Full Text PDFProc Natl Acad Sci U S A
November 2004
A gene encoding a fluorescent protein from the stony coral Lobophyllia hemprichii has been cloned in Escherichia coli and characterized by biochemical and biophysical methods. The protein, which we named EosFP, emits strong green fluorescence (516 nm) that changes to red (581 nm) upon near-UV irradiation at approximately 390 nm because of a photo-induced modification involving a break in the peptide backbone next to the chromophore. Single-molecule fluorescence spectroscopy shows that the wild type of EosFP is tetrameric, with strong Forster resonance coupling among the individual fluorophores.
View Article and Find Full Text PDFWe screened nonbioluminescent, azooxanthellate cnidaria as potential sources for advanced marker proteins and succeeded in cloning a tetrameric green fluorescent protein (GFP) from the tentacles of Cerianthus membranaceus. The fluorescence of this protein (cmFP512) is characterized by excitation maximum at 503 nm, emission maximum at 512 nm, extinction coefficient of 58,800 M-1 cm-1, quantum yield of 0.66, and fluorescence lifetime of 2.
View Article and Find Full Text PDFRed fluorescent proteins are important tools in fluorescence-based life science research. Recently, we have introduced eqFP611, a red fluorescent protein with advantageous properties from the sea anemone Entacmaea quadricolor. Here, we have studied the submillisecond light-driven intramolecular dynamics between bright and dark states of eqFP611 and, for comparison, drFP583 (DsRed) by using fluorescence correlation spectroscopy on protein solutions.
View Article and Find Full Text PDFLik Sprava
September 2000
Basic metabolic pathways were studied of formation of the adaptive syndrome in the organism of patients with grave gestoses: glycolysis, gluconeogenesis, and pentosephosphate pathway of production of nicotinamide coenzymes. It has been found out that a stressful character of reconstruction of metabolic homeostasis tends to change the processes of glycolysis and gluconeogenesis that had come to be formed by evolution. This warrants further study, its purpose being a specific correction of intracellular metabolism and prevention of complications.
View Article and Find Full Text PDFThe role has been studied of thiol compounds as indicators of bodily antioxidant defence in patients with severe gestoses. A comprehensive evaluation was done of disorders in the system lipid peroxidation/antioxidant defence, with effectiveness of their correction with ozonohemotherapy and antioxidants included into the conventional intensive therapy of patients having been studied as well. The above therapy makes for restoration of intracellular environment of erythrocytes at the expense of maintenance and augmentation of the compensatory reaction in respect of activation of the enzymic link of the antioxidant defense in blood plasma of patients.
View Article and Find Full Text PDFThe content was studied of the endogenous intoxication syndrome markers (medium-size molecules) in red cells, blood plasma and urine of patients with grave gestoses in view of the fact that underestimation of the syndrome considered tends to significantly narrow the broadness of pathogenetic notions about the given pathology and to negatively influence the effectiveness of intensive therapy of patients. Besides, the problem gets more complicated because of pathogenetic specificities of severe gestoses (the use of the well-known methods of detoxication is made difficult in the patients). Ozone hemotherapy as an efferent method of detoxication and employment of antioxidant in the complex of intensive therapy of patients with severe gestoses make for lowering the degree of catabolic disturbances by decreasing the level of medium-size molecules in erythrocytes, blood plasma and urine.
View Article and Find Full Text PDFThe paper is concerned with disturbances in major metabolic routes of bodily energy supply in patients with gestoses, such as glycolysis, gluconeogenesis, and pentosephosphate route of production of nicotinamide coenzymes. Dysfunctional breakages in the mechanisms of homeostasis maintenance, first of all, of adaptation systems in the obstetric pathology under consideration are multiform and interrelated. In addition, pathogenetical specificity of gestoses necessitates a specific correction of intracellular metabolism in order that complications might be prevented.
View Article and Find Full Text PDFThe paper is concerned with the study of chief markers of endogenous intoxication such as bodily content of medium-weight molecules (MWM) in patients with grave forms of gestoses, as well as with quest for ways of initiating a correcting therapy. Results of complex studies permitted coming to the conclusion that endogenous intoxication in different forms of gestoses runs its course through stages. This validates not only MWM levels but peculiarities of their distribution in erythrocytes, blood plasma and urine of patients as well.
View Article and Find Full Text PDFA study made on biochemical mechanisms of metabolic abnormalities in grave forms of gestosis should allow some judgement about gravity of pathophysiological alterations developing in the patients' organism, efficiency of intensive therapy, and also attest to the need for instituting some additional corrective measures. Measured in the study were the principle indices for bodily metabolic homeostasis (LPO intensity, AOD, thiol metabolism) in physiological pregnancy and labor as well as in grave forms of gestoses (preeclampsia, gestational hypertension, eclampsia). The obtained results indicated that it is necessary that a specific correction of intracellular metabolism should be carried out in order that possible complications might be prevented.
View Article and Find Full Text PDFChanges in indices for the principle metabolic pathways of bodily adaptation in gestoses were studied as were potentialities of correction thereof with ozonohemotherapy. Revealed in particular was an indirect influence of OHT on glycolysis and gluconeogenesis together with a stimulatory action on the activity of the key enzyme of the pentosephosphatic pathway. Disturbances in the course of processes of glycolysis and gluconeogenesis in the organism of the gestoses patients warrant further study to work out options for a specific correction of intracellular metabolism in the given pathology and to prevent complications.
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