Miguel Berrios

DNA from Drosophila melanogaster β-heterochromatin binds specifically to nuclear lamins in vitro and the nuclear envelope in situ

A DNA fragment designated lambda 20p1.4 binds in vitro to polymerized Drosophila melanogaster lamin. In situ hybridization of lambda 20p1.4 to isolated polytene chromosomes revealed localization at the chromocenter and to the 49 CD region on the right arm of chromosome 2. About 120 copies of sequences homologous to lambda 20p1.4 were detected per haploid genome. Nucleotide (nt) sequence analysis demonstrates that lambda 20p1.4 is an A + T-rich, 1327-bp fragment containing four repeated units between nt 595 and 919. Results suggest that lamin interacts with a region of lambda 20p1.4 between nt 300 and 1000. Confocal immunofluorescence co-localization demonstrates that in situ, the major locus of lambda 20p1.4 hybridization, the chromocenter, is found juxtaposed to the nuclear envelope (lamina). This is the first demonstration that a DNA sequence that binds specifically to nuclear lamins in vitro, is located at or near the nuclear envelope in situ and, presumably, in vivo.

Receptor density and cAMP accumulation: Analysis in CHO cells exhibiting stable expression of a cDNA that encodes the beta2-adrenergic receptor

The relationship between hormone receptor number and hormone-stimulated cAMP accumulation was probed in CHO cells that were transfected with the cDNA encoding the beta-adrenergic receptor under the control of the SV40 early promoter (expression vector pSV2BAR). CHO cells were cotransfected with pSV2BAR and expression vector pHOMER that directs the expression of a neomycin-resistance gene, and stable transfectants were selected. Clones expressing receptor at levels from 30 (wild-type) to 6000 fmol/mg membrane protein were isolated and further characterized for receptor mRNA content (measured by solution hybridization with a single-stranded cDNA probe), steady-state expression of receptor (measured by immunoblotting and indirect immunofluorescence), and their ability to accumulate intracellular cAMP in response to a beta-adrenergic agonist. Receptor mRNA content and the steady-state level of receptor protein and its expression at the cell surface were found to increase with receptor density as measured by radioligand binding. Over a 200-fold range of receptor expression, CHO transfectants displayed increasing efficacy of agonist-stimulated cAMP accumulation and increasing maximal cAMP accumulation in response to agonist. These data provide for the first time an analysis of the relationship between the density of a G-protein-linked receptor and a receptor-mediated response under conditions where the levels of G-proteins and adenylate cyclase are unaltered.

Nuclear formation in a Drosophila cell-free system.

A cell-free preparation obtained from 0- to 5-h-old Drosophila melanogaster embryos induces chromatin decondensation and nuclear formation from demembranated Xenopus sperm. Newly formed nuclei have a peripheral lamina, a double membrane, and structures resembling pore complexes. Indirect immunofluorescence analyses demonstrate the association of Drosophila lamins and DNA topoisomerase II with newly assembled nuclei.

Light-induced proteolysis of myosin heavy chain by Rose Bengal-conjugated antibody complexes

The specific light-induced, non-enzymatic digestion of chicken skeletal muscle myosin heavy chain by xanthene dye-conjugated antibodies is reported. The xanthene dye Rose Bengal was conjugated to either a mouse monoclonal anti-myosin primary specific antibody or to goat anti-mouse IgG secondary antibodies. Under our experimental conditions, visible light induced the non-enzymatic breakdown of myosin heavy chains when chicken skeletal muscle myosin either directly formed a complex with Rose Bengal-conjugated anti-myosin antibodies or indirectly formed a complex with anti-myosin antibody-Rose Bengal-conjugated secondary antibodies. The rate of the photochemical reaction depended on irradiation time and temperature. Although SDS-PAGE and immunoblot analyses showed that fragments migrating below the myosin heavy chain polypeptide predominated, these analyses also showed higher molecular mass polypeptides were generated.

The biology of β-adrenergic receptors: Analysis in human epidermoid carcinoma A431 cells

1. G-protein-linked transmembrane signaling has emerged as a major pathway for information transduction across the cell membrane. 2. In addition to photopigments that propagate the signal from light, cell-surface receptors for hormones, neurotransmitters, and autacoids propagate signals from ligand binding to membrane-bound effector units via G-proteins. 3. Biochemical and molecular features of one prominent member of these receptors, the beta-adrenergic receptor, will be highlighted in the present article. 4. The role of the human epidermoid carcinoma A431 cells as a model for the study of the structure and biology of beta-adrenergic receptors will be emphasized. 5. A model for receptor regulation, gleaned from recent advances in the biochemistry, cell and molecular biology of beta-adrenergic receptors, is discussed.

Intranuclear distribution of DNA topoisomerase II and chromatin.

Domain-specific anti-Drosophila DNA topoisomerase II antibodies were generated, affinity purified and used for confocal laser scanning immunofluorescence microscopy. Except for the nucleolus, DNA topoisomerase II is distributed throughout interphase nuclei. In adult accessory glands as well as third instar larval neural ganglion and imaginal disk nuclei, DNA topoisomerase II shows areas of co-localization with chromatin adjacent to areas of extrachromosomal distribution. These observations made in a variety of tissues under different fixation conditions and with a number of molecular probes support the notion that DNA topoisomerase II is a component of a substantially extrachromosomal network that functions to organize interphase chromatin within nuclei.

Antifading agents for confocal fluorescence microscopy.

Publisher Summary The chapter discusses strategies to reduce specimen-associated background fluorescence and the properties and performance of several antifading agents during the acquisition of multiple optical sections. Discussions are focused on indirect immunofluorescence, and other in situ localization methods are also discussed. Currently, single- and dual labeling immunofluorescence, imaging of green fluorescent protein, and fluorescent in situ hybridization (FISH) are the most widely used applications of the confocal laser scanning microscope (CLSM). Single- or dual-labeling immunofluorescence may involve either a direct or an indirect method. The greatest advantage of the CLSM is its ability to discriminate between signals originating from in-focus and out-of-focus optical planes to produce digital images that can be merged pixel by pixel to generate perfectly registered two- and three-dimensional renditions. The chapter also discusses specimen preparation, antifading agents, and acquisition of fading fluorescent images. The reduction of specimen-associated background fluorescence is performed immediately after paraformaldehyde fixation of the sample.

Anti-Fading Agents for Confocal Immunofluorescence: Colocalization of Nuclear Polypeptides

We evaluated the performance of four anti-fading agents during acquisition of multiple optical sections near the widest diameter of Drosophila accessory gland nuclei using indirect immunofluorescence and confocal laser scanning microscopy. Two commercially available agents, Vectashield and SlowFade showed anti-fading properties that alleviated fluorochrome fading associated with the acquisition of multiple fluorescent optical Z-series from a single specimen by a confocal laser scanning system. Using these reagents, we were able to colocalize polypeptides through immunostained whole Drosophila nuclei.

Oxidation of guanine in liver and lung DNA of prematurely aging OXYS rats

Immunofluorescence assay was applied for determination of 8-oxoguanine (8-oxoG) in DNA. The 8-oxoG content in liver and lung DNA of 2-and 18-month-old Wistar rats was compared with that of prematurely aging OXYS rats. It was shown that for rats of both strains, 8-oxoG content in lung DNA compared with liver DNA was 1.7–2.0-fold and 1.3–1.7-fold higher for 2-and 18-month-old rats, respectively. However, the degree of oxidative damage in liver DNA of OXYS rats was 2.4-(p < 0.01) and 1.5-fold (p < 0.05) higher for 2-and 18-month-old animals, respectively, than that in liver DNA of Wistar rats. Oxidation of guanine in lung DNA of OXYS rats was 2-(p < 0.01) and 1.7-fold (p < 0.05) higher for 2-and 18-month-old animals, respectively, than that in lung DNA of Wistar rats. The data indicate that elevated DNA oxidative damage in various organs of OXYS rats may be an important factor of accelerated again and progression of age-related diseases—cataract, macular dystrophy, hypertension, osteoporosis, cognitive and behavioral dysfunctions, and also lung and liver pathologies.

Site-directed photochemical disruption of the actin cytoskeleton by actin-binding Rose Bengal-conjugates

The in situ light-induced, non-enzymatic digestion of cytoskeletal actin by a xanthene dye conjugated to heavy meromyosin, anti-actin antibodies and/or anti-myosin antibodies is reported. The dye Rose Bengal was conjugated to either anti-actin antibodies, anti-myosin antibodies or heavy meromyosin. Under our experimental conditions, visible light induced the non-enzymatic breakdown of cytoskeletal actin when mammalian tissue culture cells were probed either with Rose Bengal-conjugated anti-actin and/or anti-myosin antibodies. Similar results were obtained when tissue culture cells were probed with Rose Bengal-conjugated heavy meromyosin before irradiation with visible light. The in situ photochemical reaction depended on the presence of actin-binding Rose Bengal-conjugates.