Sulforaphane enhances progerin clearance in Hutchinson–Gilford progeria fibroblasts

Hutchinson–Gilford progeria syndrome (HGPS, OMIM 176670) is a rare multisystem childhood premature aging disorder linked to mutations in the LMNA gene. The most common HGPS mutation is found at position G608G within exon 11 of the LMNA gene. This mutation results in the deletion of 50 amino acids at the carboxyl‐terminal tail of prelamin A, and the truncated protein is called progerin. Progerin only undergoes a subset of the normal post‐translational modifications and remains permanently farnesylated. Several attempts to rescue the normal cellular phenotype with farnesyltransferase inhibitors (FTIs) and other compounds have resulted in partial cellular recovery. Using proteomics, we report here that progerin induces changes in the composition of the HGPS nuclear proteome, including alterations to several components of the protein degradation pathways. Consequently, proteasome activity and autophagy are impaired in HGPS cells. To restore protein clearance in HGPS cells, we treated HGPS cultures with sulforaphane (SFN), an antioxidant derived from cruciferous vegetables. We determined that SFN stimulates proteasome activity and autophagy in normal and HGPS fibroblast cultures. Specifically, SFN enhances progerin clearance by autophagy and reverses the phenotypic changes that are the hallmarks of HGPS. Therefore, SFN is a promising therapeutic avenue for children with HGPS.     

Effect of dietary sodium on vasoconstriction and eNOS-mediated vascular relaxation in caveolin-1-deficient mice

Changes in dietary sodium intake are associated with changes in vascular volume and reactivity that may be mediated, in part, by alterations in endothelial nitric oxide synthase (eNOS) activity. Caveolin-1 (Cav-1), a transmembrane anchoring protein in the plasma membrane caveolae, binds eNOS and limits its translocation and activation. To test the hypothesis that endothelial Cav-1 participates in the dietary sodium-mediated effects on vascular function, we assessed vascular responses and nitric oxide (NO)-mediated mechanisms of vascular relaxation in Cav-1 knockout mice (Cav-1-/-) and wild-type control mice (WT; Cav-1+/+) placed on a high-salt (HS; 4% NaCl) or low-salt (LS; 0.08% NaCl) diet for 16 days. After the systolic blood pressure was measured, the thoracic aorta was isolated for measurement of vascular reactivity and NO production, and the heart was used for measurement of eNOS expression and/or activity. The blood pressure was elevated in HS mice treated with NG-nitro-l-arginine methyl ester and more so in Cav-1-/- than WT mice and was significantly reduced during the LS diet. Phenylephrine caused vascular contraction that was significantly reduced in Cav-1-/- (maximum 0.25 +/- 0.06 g/mg) compared with WT (0.75 +/- 0.22 g/mg) on the HS diet, and the differences were eliminated with the LS diet. Also, vascular contraction in response to membrane depolarization by high KCl (96 mM) was reduced in Cav-1-/- (0.27 +/- 0.05 g/mg) compared with WT mice (0.53 +/- 0.12 g/mg) on the HS diet, suggesting that the reduced vascular contraction is not limited to a particular receptor. Acetylcholine (10(-5) M) caused aortic relaxation in WT mice on HS (23.6 +/- 3.5%) and LS (23.7 +/- 5.5%) that was enhanced in Cav-1-/- HS (72.6 +/- 6.1%) and more so in Cav-1-/- LS mice (93.6 +/- 3.5%). RT-PCR analysis indicated increased eNOS mRNA expression in the aorta and heart, and Western blots indicated increased total eNOS and phosphorylated eNOS in the heart of Cav-1-/- compared with WT mice on the HS diet, and the genotypic differences were less apparent during the LS diet. Thus Cav-1 deficiency during the HS diet is associated with decreased vasoconstriction, increased vascular relaxation, and increased eNOS expression and activity, and these effects are altered during the LS diet. The data support the hypothesis that endothelial Cav-1, likely through an effect on eNOS activity, plays a prominent role in the regulation of vascular function during substantial changes in dietary sodium intake.     

Receptor distribution and the mechanism of enhanced erythrocyte agglutination by soybean agglutinin

We have examined the role of receptor clustering in intact erythrocyte membranes exhibiting enhanced lectin-mediated cell agglutination by analyzing freeze-fracture and freeze-etch images of human erythrocytes labeled with ferritin-conjugated soybean agglutinin. We find that trypsinization and fixation of intact erythrocytes, in either order, causes no alteration of the random distribution of ferritin-conjugated soybean agglutinin on the surfaces of these cells as compared to their distribution on the surfaces of fixed erythrocytes and untreated erythrocyte ghosts. Furthermore, clustering of the intramembranous particles in the membrane of intact erythrocytes was not found with any of the cells described above.We conclude that clustering of the soybean agglutinin receptors is not a major factor involved in the enhanced agglutination of intact trypsinized erythrocytes. Caution is necessary in transferring information obtained with erythrocyte ghosts, where clustering can be induced, to intact erythrocytes.     

Iodine Deficiency Among Hypothyroid Patients Living in Jeddah

The objective was to determine the prevalence of iodine deficiency among hypothyroid patients and the effect of dietary goitrogens on indices of iodine and thyroid status. This is a case-control study of 106 subjects who were recruited from King Abdulaziz University Hospital, Jeddah. Blood and urine were collected for serum thyroid hormones, thyroid autoantibodies, thyroglobulin (Tg) and urinary iodine concentration (UIC). Dietary iodine and goitrogenic food intake were assessed by questionnaire. Using World Health Organization (WHO) cutoff values for UIC, both controls and cases were iodine deficient (85% and 83%, respectively). Furthermore, dietary iodine was deficient in 23% of controls and 36% of cases. In cases, there was a positive association between UIC levels and serum thyroid stimulating hormone (r = 0.405, p < 0.01) and a negative association with serum fT₄ (r = −0.358, p < 0.01). Serum Tg antibody titers were also positively associated with dietary iodine (r = 0.328, p < 0.05). Patients with elevated serum autoantibodies had lower UIC and dietary iodine than those with normal serum autoantibodies. UIC was associated with dietary goitrogens including turnip (r = 0.280, p < 0.05) and pine (r = 0.289, p < 0.05) among cases. Iodine deficiency is common and the consumption of dietary goitrogens is high among euthyroid and hypothyroid subjects living in Jeddah.     

Molecular characterization of pezizalean ectomycorrhizas associated with pinyon pine during drought

Recent studies using molecular analysis of ectomycorrhizas have revealed that ascomycete fungi, especially members of the order Pezizales, can be important members of ectomycorrhizal (EM) fungal communities. However, little is known about the ecology and taxonomy of many of these fungi. We used data collected during a wet and a dry period to test the hypothesis that pezizalean EM fungi associated with pinyon pine (Pinus edulis) responded positively to drought stress. We also assessed the phylogenetic relationships among six, unknown pezizalean EM fungi, common to our study sites, using rDNA sequences from the internal transcribed spacer and large subunit (LSU) regions of the ribosomal DNA. Sequences of these fungi were then compared to sequences from known taxa to allow finer-scale identification. Three major findings emerged. First, at two sites, pezizalean EM were 44–95% more abundant during a dry year than a wetter year, supporting the hypothesis that pezizalean EM fungi respond positively to dry conditions. Second, four of the six unknown pezizalean EM fungi associated with P. edulis separated from one another consistently regardless of site or year of collection, suggesting that they represented distinct taxa. Third, comparison with LSU sequences of known members of the Pezizales indicated that these four taxa grouped within the genus Geopora of the family Pyronemataceae. Our results provide further evidence of the importance of pezizalean fungi in the ectomycorrhizal symbiosis and demonstrate high local abundance of members of the genus Geopora in drought-stressed pinyon–juniper woodlands.     

Methylation analysis of mesophyll,epidermis, and fibre cell-walls isolated from the leaves of perennial and italian ryegrass

Intact, finely milled mesophyll, epidermis, and fibre cell-walls prepared from the leaves of perennial and Italian ryegrass have been subjected to methylation analysis. Methylation of the cell-walls led to a consistently higher recovery of glucose residues than that obtained by analysis of monosaccharide residues as their alditol acetates. Values for other sugars were in close agreement. The partially methylated sugars formed were consistent with the presence, in order of decreasing concentration, of cellulose, (glucurono)arabinoxylan, xyloglucan, rhamnogalacturonan, (1→3),(1→4)-linked glucan, (1→4)-linked galactan, and (1→3),(1→6)-linked galactan. The relative proportions of these polysaccharides differed between the various types of cell. Arabinoxylan comprised 21.6%, 26.7%, and 36.5% of the total sugars recovered from mesophyll, epidermis, and fibre cell-walls, respectively. Mixed-linked glucan and rhamnogalacturonan were found in epidermis walls in amounts 2- to 3-fold higher than in other cell-walls. The xylan backbone of arabinoxylan was more heavily substituted in primary than in secondary-thickened (fibre) cell-walls. Arabinose, found largely as terminal residues in the cell-walls, carried various amounts of alkali-labile substituents, particularly at position 5. The extent of 5-substitution reflected the phenolic content and was substantially higher in fibre cell-walls. The methylation data, coupled with the analytical data for uronic acids and non-carbohydrate components, accounted for ～98% of the cell-wall dry matter.     

Cytological effects of colchicine on the grasshopper neuroblast in vitro with special reference to the origin of the spindle

The effectiveness of colchicine in destroying or preventing the development of the spindle is determined by the concentration of the colchicine and the degree of development of the spindle at the time of exposure; the greater the concentration of colchicine, the greater will be its effectiveness in destroying the spindle or interfering with its development; the more completely the spindle is developed at the time of exposure to colchicine, the greater is the concentration of colchicine required to destroy it or prevent its further development.The series of changes in chromosome orientation that take place during the course of colchicine action, namely, approximation of centromeres, formation of “stars,” the breaking up of one “star” into several, and complete chromosome disorientation, represent successive stages in the destruction of the spindle.Destruction of the completely formed spindle is typically accompanied by the accumulation of the spindle material outside the diminishing spindle in a hyaline globule. Similarly, interference of colchicine with spindle development leads to the accumulation of the presumptive spindle material, i.e., the karyolymph, in one or more hyaline globules. It is suggested that colchicine does not destroy the spindle material but merely alters its molecular orientation, so that it comes to comprise a spherical mass with no mitotic function.Strong concentrations of colchicine cause middle and late prophase nuclei to revert to early prophase. Somewhat lower concentrations applied to late prophase nuclei occasionally delay the breakdown of the nuclear membrane without altering the rate of chromosome contraction. In greatly retarded late prophase nuclei the chromosomes lose their intranuclear orientation, which lends support to the concept that the centromeres normally maintain a fixed position within the nucleus.     

Preparation of affinity-purified,biotinylated tetanus toxin,and characterization and localization of cell surface binding sites on nerve growth factor-treated PC12 cells

Biotinylated derivatives of tetanus toxin were prepared and isolated by chromatofocusing and ganglioside-affinity chromatography. Biotinylation was monitored by the appearance of a 210,00 dalton complex upon SDS-polyacrylamide gel electrophoresis in the presence of avidin, and by selective binding to an avidin-Sepharose gel. At molar biotin:toxin ratios from 11 to 201 only biotinylated derivatives with low toxicity were obtained; these derivatives, however, retained 60–80% of their specific binding affinity for brain synaptosomes. A biotinylated tetanus toxin derivative purified by ganglioside-affinity chromatography was used to identify and localize tetanus toxin binding sites on PC12 cells. Electron microscopic analysis with streptavidin-gold revealed very low levels of tetanus toxin binding sites on the surface of untreated cells, and the appearance of such binding sites during the second week of nerve growth factor-induced differentiation. Examination of micrographs of the differentiated cells indicated that the tetanus toxin binding sites sites are concentrated on the neurites, with relatively few appearing on the cell bodies. Cognate studies using¹²⁵I-labeled, affinity-purified tetanus toxin revealed an increase in PC12 binding capacity from about 0.07 nmol/mg protein in untreated cells to 0.8 nmoles/mg protein in cells treated for 14 days with nerve growth factor. Cells treated in suspension for 2–3 weeks with nerve growth factor do not express tetanus toxin binding sites; upon plating, these cells required one week for the appearance of binding sites, although neurites grew much more rapidly from these primed cells. The high binding capacity of these tetanus toxin sites, as well as their sensitivity to neuraminidase, is indicative of a polysialoganglioside structure. The advantages of biotinylated tetanus toxin derivatives are discussed and the significance of nerve growth factor-differentiated PC12 cells grown as monolayers as a model for the study of the development, localization, and function of neuraminidase-sensitive tetanus toxin binding sites is presented.Abbreviations PBS phosphate-buffered saline - STS sucrose-Tris-serum solution - NGF nerve growth factor - C collagen - PL polylysine - BBG bovine brain ganglioside mixture - GM₁ gafactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosyl ceramide - GD_1a [N-acetylneuraminyl]-galactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosyl ceramide - GT_1a [N-aceylneuraminyl]-galactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosyl ceramide - GD_1b galactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl-N-acetylneuraminyl]-galactosylglucosyl ceramide - GT_1b [N-acetylneuraminyl]-galactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl-N-acetylneuraminyl] galactosylglucosyl ceramide - NANA N-acetylneuraminic acid     

New Polyazaporphine Chemistry for the Origin of Life. A Porphine Solar Energy Transducer for Origin of Life Chemistry: A Possible Synthetic Patj from HCN to an Octaazaporphine via 4H-Imidazol-4-Imine [(HCN)3] Related Chemistry

Molecular orbital spectral predictions suggest that 2,5,7,10,12,15,17,20-octaaza-21H, 23H-porphine has a visible spectral range closely matching that of chlorophyll-a. Since the octaazaporphine is, in its core, a simple derivative of an (HCN)₁₂ oligomer, this fact, together with its spectral properties, would suggest that it occupies a high rank as a primordial porphinic solar energy transducer for photochemistry essential to life's formation. The demonstration that the mass 324 hexahydrooctaazaporphine is formed in protic media by the cyclotetramerization of imidazol-4-aminohydroxonium ion or the derived nitrenium ion, and that a mass 318 species consonant with that of the Hückel aromatic octaazaporphine is observed in the course of these studies, strongly supports the proposed octaazaporphine synthesis in a prebiotic hydrocyanic acid milieu.     

Use of within-array replicate spots for assessing differential expression in microarray experiments

MOTIVATION: Spotted arrays are often printed with probes in duplicate or triplicate, but current methods for assessing differential expression are not able to make full use of the resulting information. The usual practice is to average the duplicate or triplicate results for each probe before assessing differential expression. This results in the loss of valuable information about genewise variability. RESULTS: A method is proposed for extracting more information from within-array replicate spots in microarray experiments by estimating the strength of the correlation between them. The method involves fitting separate linear models to the expression data for each gene but with a common value for the between-replicate correlation. The method greatly improves the precision with which the genewise variances are estimated and thereby improves inference methods designed to identify differentially expressed genes. The method may be combined with empirical Bayes methods for moderating the genewise variances between genes. The method is validated using data from a microarray experiment involving calibration and ratio control spots in conjunction with spiked-in RNA. Comparing results for calibration and ratio control spots shows that the common correlation method results in substantially better discrimination of differentially expressed genes from those which are not. The spike-in experiment also confirms that the results may be further improved by empirical Bayes smoothing of the variances when the sample size is small. AVAILABILITY: The methodology is implemented in the limma software package for R, available from the CRAN repository http://www.r-project.org