Nrl and Sp nuclear proteins mediate transcription of rod-specific cGMP-phosphodiesterase beta-subunit gene: involvement of multiple response elements.

cGMP-phosphodiesterase (PDE) is the key effector in rod photoreceptor signal transduction. Mutations in the gene encoding its catalytic beta-subunit (beta-PDE) cause retinal degenerations leading to blindness. We report that the short -93 to +53 sequence in the upstream region of this gene is sufficient for beta-PDE transcription in both Y79 human retinoblastoma cells and Xenopus embryo heads maintained ex vivo. This sequence also functions as a minimal rod-specific promoter in transgenic Xenopus tadpoles. The Nrl transcription factor binds in vitro to the betaAp1/NRE regulatory element located within this region and transactivates it when overexpressed in nonretinal 293 embryonic kidney cells. We also found a G/C-rich activator element, beta/GC, important for promoter activity in Y79 retinoblastoma cells and Xenopus embryos. Both the ubiquitous Sp1 and the central nervous system-specific Sp4 transcription factors are expressed in retina and interact with this element in vitro. Electrophoretic mobilities of beta/GC-Y79 nuclear protein complexes are altered by antibodies against Sp1 and Sp4. Thus, our results implicate Nrl, Sp1, and Sp4 in transcriptional regulation of the rod-specific minimal beta-PDE promoter. We also conclude that Xenopus laevis is an efficient system for analyzing the human beta-PDE promoter and may be used to study other human retinal genes ex vivo and in vivo.

Transient transfection of human retinoblastoma cells : application to the analysis of the regulatory regions of photoreceptor-specific genes.

Retinoblastoma (Rb) is an intraocular tumor usually diagnosed in children under four years of age (1). The tumor rises when both alleles of the Rb tumor suppressor gene become inactivated in a retinal precursor cell during development (2,3). The first retinoblastoma cell line to be established in culture, Y-79 (4), has been shown to originate from neuroectodermal cells that express both neuronal and glial cell markers (3). Both Y-79 cells and Rb tumor cells produce mRNAs encoding several proteins unique to the photoreceptors (5), including different subunits of cone- and rod-specific cGMP-phosphodiesterases (6). Therefore, cultured Y-79 cells, which have a human retinal origin, could be particularly useful for studying the regulatory mechanisms of photoreceptor-specific gene expression (7).

Transcriptional activation of the human rod cGMP-phosphodiesterase beta-subunit gene is mediated by an upstream AP-1 element.

During photoactivation retinal cGMP-phosphodiesterase (PDE) mediates signal transduction in the photoreceptor outer segments. Mutations in the beta-subunit gene of rod-specific PDE (beta-PDE) have been associated with inherited retinal degeneration in a number of species, including human. Here we have investigated the proximal upstream sequences that participate in transcriptional activation of this gene. Transient transfections demonstrated that the sequence from -72 to +53 bp contained sufficient information to direct high levels of gene expression in cells of retinal origin. Deletion or mutagenesis of an AP-1 motif present in this region caused 90-95% reduction in reporter gene expression. By gel mobility shift assay we demonstrated specific interactions between putative nuclear transcription factors and this AP-1 element. These findings indicate that the proximal AP-1 site in the human beta-PDE promoter is functionally relevant and necessary for transcriptional activation of this gene.

Solution dynamics of the 1,2,3,4,6-penta-O-acetyl-alpha-D-idopyranose ring.

The anticoagulant properties of heparin are thought to derive from the inhibition of thrombin and other coagulation-related proteases by the binding of heparin to cofactors such as antithrombin III and heparin cofactor II. The apparent minimum native heparin sequence which can bind to antithrombin III is a highly sulfated pentasaccharide which contains a 2-O-sulfo-alpha-L-idopyranosyluronic acid residue. The idopyranosyl residue has the unusual property of existing in the solution state as a mixture of ring conformers. Whereas most hexopyranose sugars exist as a single chair conformer (eg D-glucose exists overwhelmingly as a (4)C1 chair), the idopyranosyl ring is known to rapidly exchange between at least two and often more distinct conformations, depending on type and number of substituents (hydroxyl, carboxyl, sulfate, etc.) and solvent conditions (solvent pH, salt concentration, temperature). It is believed that this flexibility of the idopyranosyl residue in heparin is related to its binding specificity. In the past, coupling constants and molecular dynamics have been used to estimate the relative populations of conformers in iduronate and related compounds. Here we report extensive NMR measurements, including line shape analysis, T1p measurements, T1 and NOE measurements and spectral density mapping, which have been used to study the dynamics of conformer interconversion in model compounds related to idose and glucose. The findings presented here indicate that 1,2,3,4,6-penta-O-acetyl-alpha-D-idopyranose can be reasonably well described as existing in a two-state equilibrium consisting of the (4)C1 and (0)S2 conformers. (13)C NMR line shape analysis yields a deltaH+ of 40 kJ mol(-1) and a deltaS++ of 31 J mol(-1) K(-1) for the (4)C1 --> (0)S2 interconversion and a deltaH++ of 31 kJ mol(-1) and a deltaS++ of 13 J mol(-1) K(-1) for the (0)S2 --> (4)C1 interconversion. This corresponds to exchange rates of 22 and 128 MHz, respectively, at room temperature.

Hyaluronan in the human trabecular meshwork.

PURPOSE: Hyaluronan (HA) is a high-molecular weight glycosaminoglycan that can affect water and solute fluxes in the extracellular matrix. The distribution of HA in the human trabecular meshwork of nonglaucomatous eyes was examined to help understand the potential role of HA in the regulation of aqueous outflow resistance. METHODS: Histolocalization of HA was established in situ in the trabecular meshwork of human eyes with no known diseases of the anterior segment. A specific biotinylated HA-binding peptide was used as a probe for this study, with enhanced sensitivity of HA detection achieved by modifications of the fixation and staining procedures. RESULTS: Evaluation of HA staining in the aqueous outflow pathway in comparison to that in other ocular structures (e.g., the vitreous) showed pronounced staining in the trabecular meshwork. The staining intensity was similar between various layers of the meshwork. Both the filtering and the anterior nonfiltering portions of the trabecular meshwork showed pronounced HA staining. The staining was localized primarily to the trabecular meshwork endothelial cells. CONCLUSIONS: Pronounced HA staining observed in the various layers of the trabecular meshwork suggests that substantial amount of HA is present in the nonglaucomatous outflow pathway. The staining pattern suggests that HA is associated with the endothelial cells lining the trabecular beams. This finding supports potential roles for this glycosaminoglycan in the regulation of the physiological aqueous outflow resistance or in the maintenance of the outflow channels or both. Histochemical localization of HA in the various layers of the non-glaucomatous meshwork provides a useful basis for future comparative studies of HA distribution and relative amounts in the trabecular meshworks of eyes affected by various types of glaucoma.

A comparison of the conformation of sucrose octasulfate, free and bound to acidic fibroblast growth factor.

The conformation of sucrose octasulfate free in solution has been determined based on high-resolution NMR spectroscopy. Three-bond 1H-1H scalar coupling constants, laboratory and rotating frame NOEs, long-range 1H-13C scalar coupling constants, and chemical-shift temperature and ionic-strength dependence were used, aided by molecular mechanics calculations. By modification of a pulse sequence designed for measuring long-range 1H-13C coupling constants, it was possible to obtain an accurate value for the 3JC2f-H1g despite the confounding presence of 3JHH of similar value. Free sucrose octasulfate appears to assume a conformation significantly different from any of the eight conformations observed bound to acidic fibroblast growth factor, as determined in a previous X-ray crystallographic study [X. Zhu, B.T. Hsu, and D.C. Rees, Structure, 1 (1993) 27-34]. Strong electrostatic interactions between guest and host may be the dominant factor in deformation of sucrose octasulfate. The implications of this study for protein-carbohydrate interactions and the effects of the presence of sulfate groups on the flexibility of sucrose are also discussed.

The solution conformation of hyaluronan: a combined NMR and molecular dynamics study.

Hyaluronan (HA) is a negatively charged glycosaminoglycan that exhibits a wide variety of biological effects mediated by binding to cell-surface and extracellular matrix proteins (hyaladherins). Short HA oligosaccharides have been shown to retain the specific interactions and biological effects of high molecular weight HA. Although it has a simple disaccharide repeating unit, the aqueous solution conformation of HA has been very difficult to determine because of strong coupling and overlapping resonances. In this study, we propose aqueous solution conformations for an octasaccharide of HA, derived from proton-proton NOE data and restrained molecular dynamics. To overcome spectral overlap and strong coupling, alternate methods for extracting distance restraints were employed. Restrained molecular dynamics calculations yielded one set of interglycosidic angle values for the beta (1,3) linkage (phi 13 = 46 degrees, psi 13 = 24 degrees). In contrast, two sets of values for the beta (1,4) linkage were consistent with the NOE restraints (phi 14 = 24 degrees, psi 14 = -53 degrees or phi 14 = 48 degrees, psi 14 = 8 degrees). The potential difference in flexibility for the two linkages is consistent with unrestrained as well as the restrained molecular dynamics trajectories described here. The conformational parameters obtained from restrained molecular dynamics are used to predict helical parameters of high molecular weight HA and will provide a basis for studies of HA binding to proteins.

Magnesium-DNA interactions from interpretation of 25Mg-nmr relaxation rates: field and coion dependence.

We have used 25Mg-nmr to investigate the binding of magnesium ions to double-stranded DNA. We have measured line shapes for 25Mg in the presence of monodisperse calf thymus DNA (160 base pairs; b.p.) (magnesium: phosphate = 2.0) at two different field strengths, 11.75 T and 7.05 T, and used the isotropic model of two-site exchange developed by Westlund and Wennerstrom to simultaneously fit the line shapes at both field strengths. This model does not reproduce the observed field dependence. This is in contrast to a previous study [E. Berggren, L. Nordenskiold, and W.H. Braunlin (1992), Biopolymers, Vol. 32, pp. 1339-1350] in which a similar model of isotropic two-site exchange qualitatively reproduced the temperature dependence of the line widths. Relaxation rates were also measured as a function of magnesium: phosphate ratio and coion type. These measurements were used to assess the sensitivity of magnesium relaxation measurements to small changes in DNA structure induced by changes in the solvent environment. The temperature dependence of the line shape varies with the type of coion (chloride or sulfate) present. This coion dependence of the line shape is consistent with the coion dependence of the aggregation midpoint temperature reported by Bloomfield and co-workers [O.A. Knoll, M.G. Fried, and V.A. Bloomfield (1988) in Structure and Expression, Vol. 2, R.H. Sarma and M. H. Sarma, Eds., Adenine Press, New York] and attributed to a lyotropic effect. These results suggest that even at low magnesium: phosphate ratios, relaxation parameters are specific to each magnesium-coion-DNA system.

NMR studies of peptides derived from the putative binding regions of cartilage proteins. No evidence for binding to hyaluronan.

Previous work has implicated sequences within the tandem repeats of cartilage link protein in the interaction of link protein with hyaluronan. This conclusion was based on competitive inhibition experiments using synthetic peptides (Goetinck, P. F., Stirpe, N. S., Tsonis, P. A., and Carlone, D. (1987) J. Cell Biol. 105, 2403-2407). Further investigation of this system using high resolution nuclear magnetic resonance, circular dichroism, and competitive inhibition with other peptides indicates that the previously observed inhibition of link protein-hyaluronan binding was not caused by peptide-hyaluronan interactions. Instead, nonspecific aggregation of the peptides with link protein is proposed to account for all of the experimental data. Consequently, there is no direct experimental evidence to support the conclusion that these sequences in the tandem repeats of link protein are responsible for the link protein-hyaluronan interaction. If these peptides do represent the hyaluronan binding regions of link protein, these results imply a highly structure-dependent interaction between link protein and hyaluronan. Conformational analysis of the peptides using two-dimensional nuclear Overhauser spectroscopy indicates that the linear peptides do not adopt any stable secondary structure. However, several residues in the disulfide-looped peptides exhibit connectivities, suggesting a relatively long-lived extended chain conformation, consistent with predictions of secondary structure based on sequence analysis.

[Nutrient media for fermenting Actinomyces]. / Pitatel'nye sredy dlia fermentiruiushchikh aktinomitsetov.

Fitness of various commercial nutrient media, manufactured in the USSR, for isolation and cultivation of fermentation-producing actinomyces was under study. The media were tested in two stages. Reference and newly isolated actinomyces cultures were examined at stage 1, clinical material at stage 2. Hottinger's blood agar was found the best for maintaining the growth of facultative anaerobic actinomyces and possessed the highest differentiating characteristics. This medium maintained sufficiently intensive growth of anaerobic actinomyces but only in the presence of meat extract. A liquid nutrient medium was designed, based on Hottinger's hydrolysate, that may be used for studies of these microorganisms' physiologic parameters.