Comparative assessment of fluorescent transgene methods for quantitative imaging in human cells

Fluorescence tagging of proteins is a widely used tool to study protein function and dynamics in live cells. However, the extent to which different mammalian transgene methods faithfully report on the properties of endogenous proteins has not been studied comparatively. Here we use quantitative live-cell imaging and single-molecule spectroscopy to analyze how different transgene systems affect imaging of the functional properties of the mitotic kinase Aurora B. We show that the transgene method fundamentally influences level and variability of expression and can severely compromise the ability to report on endogenous binding and localization parameters, providing a guide for quantitative imaging studies in mammalian cells.     

Structures of adamalysin II with peptidic inhibitors. Implications for the design of tumor necrosis factor alpha convertase inhibitors.

Crotalus adamanteus snake venom adamalysin II is the structural prototype of the adamalysin or ADAM family comprising proteolytic domains of snake venom metalloproteinases, multimodular mammalian reproductive tract proteins, and tumor necrosis factor alpha convertase, TACE, involved in the release of the inflammatory cytokine, TNFalpha. The structure of adamalysin II in noncovalent complex with two small-molecule right-hand side peptidomimetic inhibitors (Pol 647 and Pol 656) has been solved using X-ray diffraction data up to 2.6 and 2.8 A resolution. The inhibitors bind to the S'-side of the proteinase, inserting between two protein segments, establishing a mixed parallel-antiparallel three-stranded beta-sheet and coordinate the central zinc ion in a bidentate manner via their two C-terminal oxygen atoms. The proteinase-inhibitor complexes are described in detail and are compared with other known structures. An adamalysin-based model of the active site of TACE reveals that these small molecules would probably fit into the active site cleft of this latter metalloproteinase, providing a starting model for the rational design of TACE inhibitors.     

Heterogeneity of mammalian DNA ligase detected on activity and DNA sequencing gels.

A new method to detect DNA ligase activity in situ after NaDodSO4 polyacrylamide gel electrophoresis has been developed. After renaturation of active polypeptides the ligase reaction occurs in situ by incubating the intact gel in the presence of Mg++ and ATP. Further treatment with alkaline phosphatase removes the unligated 5'-32P-end of oligo (dT) used as a substrate and active polypeptides having ligase activity are identified by autoradiography. Analysis on DNA sequencing gels of the oligo (dT) reaction products present in the activity bands ensures that the radioactive material detected in activity gels or in standard in vitro ligase assays corresponds unambiguously to a ligase activity. Using these methods, we have analysed the purified phage T4 DNA ligase, and the activities present in crude extracts and in purified fractions from monkey kidney (CV1-P) cells. The purified T4 enzyme yields one or two active peptides with Mr values of 60,000 and 70,000. Crude extracts from CV1-P cells contain several polypeptides having DNA ligase activity. Partial purification of these extracts shows that DNA ligase I isolated from hydroxylapatite column is enriched in polypeptides with Mr 200,000, 150,000 and 120,000, while DNA ligase II is enriched in those with Mr 60,000 and 70,000.     

Extremely thermostable glutamate dehydrogenase from the hyperthermophilic archaebacterium Pyrococcus furiosus.

The hyperthermophilic archaebacterium Pyrococcus furiosus contains high levels of NAD(P)-dependent glutamate dehydrogenase activity. The enzyme could be involved in the first step of nitrogen metabolism, catalyzing the conversion of 2-oxoglutarate and ammonia to glutamate. The enzyme, purified to homogeneity, is a hexamer of 290 kDa (subunit mass 48 kDa). Isoelectric-focusing analysis of the purified enzyme showed a pI of 4.5. The enzyme shows strict specificity for 2-oxoglutarate and L-glutamate but utilizes both NADH and NADPH as cofactors. The purified enzyme reveals an outstanding thermal stability (the half-life for thermal inactivation at 100 degrees C was 12 h), totally independent of enzyme concentration. P. furiosus glutamate dehydrogenase represents 20% of the total protein; this elevated concentration raises questions about the roles of this enzyme in the metabolism of P. furiosus.     

Synthesis and secretion of interphotoreceptor retinoid-binding protein (IRBP) by isolated normal and rd mouse retinal photoreceptor neurons in culture

Cultures of dissociated retinal neurons and photoreceptors from homozygous wild-type, heterozygous rd/+ and homozygous rd/rd retinas have been used to investigate the capacity of isolated photoreceptor cells to synthesize and secrete the interphotoreceptor retinoid-binding protein (IRBP). Retinal cells were dissociated on postnatal day 2 and grown in chemically defined medium in the absence of glial and pigmented epithelial cells. Expression of IRBP immunoreactive materials in these cultures was cell type-specific and developmentally regulated. Thus increasing numbers of rod photoreceptor cells showed immunoreactivity during the first week in culture, whereas nonphotoreceptor cell types remained consistently negative. Photoreceptor immunoreactivity could be detected in permeated (detergent-treated) as well as in unpermeated preparations, the latter suggesting that some IRBP is associated with the photoreceptor cell surface. These materials appeared to be loosely bound to the photoreceptors, since they disappeared when the cultures were exposed for 6 hr to IRBP-free medium but not when they were exposed to IRBP-containing medium. IRBP synthesis and secretion could be demonstrated by analyzing either cell extracts or conditioned medium by "slot blot" and Western blot techniques using affinity purified antibodies against bovine IRBP as well as by fluorographic analysis after metabolic labeling of the cultures with 35S-methionine. Comparisons of cultures from the different genotypes showed many similarities, including the abundance of IRBP-immunoreactive photoreceptors in 7 day cultures. However, immunochemical analysis showed lower conditioned medium/cell extract IRBP ratios in rd/rd cultures, an observation consistent with previous reports suggesting that IRBP secretion may be deficient in rd/rd photoreceptor cells.     

Myocardial Dysfunction Induced by Food Restriction is Related to Morphological Damage in Normotensive Middle-Aged Rats

Summary Previous works from our laboratory have revealed that food restriction (FR) promotes discrete myocardial dysfunction in young rats. We examined the effects of FR on cardiac function, in vivo and in vitro, and ultrastructural changes in the heart of middle-aged rats. Twelve-month-old Wistar-Kyoto rats were fed a control (C) or restricted diet (daily intake reduced to 50% of the control group) for 90 days. Cardiac performance was studied by echocardiogram and in isolated left ventricular (LV) papillary muscle by isometric contraction in basal condition, after calcium chloride (5.2 mM) and beta-adrenergic stimulation with isoproterenol (10⁻⁶ M). FR did not change left ventricular function, but increased time to peak tension, and decreased maximum rate of papillary muscle tension development. Inotropic maneuvers promoted similar effects in both groups. Ultrastructural alterations were seen in most FR rat muscle fibers and included, absence and/or disorganization of myofilaments and Z line, hyper-contracted myofibrils, polymorphic and swollen mitochondria with disorganized cristae, and a great quantity of collagen fibrils. In conclusion, cardiac muscle sensitivity to isoproterenol and elevation of extracellular calcium concentration is preserved in middle-aged FR rats. The intrinsic muscle performance depression might be related to morphological damage.     

In vitro antioxidant activities of antioxidant-enriched toothpastes

Several forms of periodontal diseases (PD) are often associated with modified phagocytosing leukocytes and contemporary free radical production. Host antioxidant defenses could benefit from toothpastes used as adjuncts to counteract plaque-associated bacteria. The aim of the present study was to determine possible antioxidant activity (AA) of 12 differently antioxidant-enriched toothpastes, regardless of their efficacy as antimicrobial agents. Toothpastes were enriched alternatively with sodium ascorbyl phosphate, alpha-tocopherol acetate, pycnogenol, allantoin and methyl salycilate or a mixture of these. AA was tested in a cell-free system with a ABTS-decolorization assay improved by means of a flow injection analysis device. Comet assay, using NCTC 2544 keratinocytes, was performed to test if it was possible to identify any protection against in vitro DNA fragmentation provoked by a challenge with H(2)O(2) in cultures pre-incubated with toothpaste extracts. Only toothpastes containing sodium ascorbyl phosphate displayed clear AA with I(50) values ranging between 50 and 80 mg of toothpaste/ml water. COMET analysis of cells challenged with H(2)O(2) in presence of toothpaste extracts revealed a limited protection exerted by sodium ascorbyl phosphate. The results described herein indicate that toothpastes containing sodium ascorbyl phosphate possess AA. All the data were obtained in systems in vitro and the demonstration of in vivo AA is desirable. These findings could be useful in the treatment and maintenance of some forms of PD and should be considered when arranging new toothpaste formulations.     

Oligomerization of Sulfolobus solfataricus signature amidase is promoted by acidic pH and high temperature

The recombinant amidase from the hyperthermophylic archaeon Sulfolobus solfataricus (SSAM) a signature amidase, was cloned, purified and characterized. The enzyme is active on a large number of aliphatic and aromatic amides over the temperature range 60-95 degrees C and at pH values between 4.0 and 9.5, with an optimum at pH 5.0. The recombinant enzyme is in the form of a dimer of about 110 kD that reversibly associates into an octamer in a pH-dependent reaction. The pH dependence of the state of association was studied using gel permeation chromatography, analytical ultracentrifugation and dynamic light scattering techniques. At pH 7.0 all three techniques show the presence of two species, in about equal amounts, which is compatible with the existence of a dimeric and an octameric form. In decreasing pH, the dimers formed the octameric species and in increasing pH, the octameric species was converted to dimers. Above pH 8.0, only dimers were present, below pH 3.0 only octamers were present. The association of dimers into octamers decreased in non-polar solvents and increased with temperature. A mutant (Y41C) was obtained that did not show this behavior.