Investigation of the effects of antisense oligodeoxynucleotides to islet amyloid polypeptide mRNA on insulin release, content and expression

We have investigated the effects of antisense oligodeoxynucleotides (oligos) to islet amyloid polypeptide (IAPP) mRNA on the expression and secretion of IAPP and insulin, in the clonal beta-cell line HIT-T15. Phosphorothioate-modified oligos were cytotoxic compared with phosphodiester (D)-oligos. Of the nine oligos tested using a lipofection reagent, O3, a 30-mer D-oligo complementary to a sequence downstream of the IAPP initiation codon, showed a significant dose-dependent suppression of IAPP mRNA, with a 42% decrease at 7.5 microM, compared with a scrambled (MSO3) control oligo (n = 3, P < 0.01). A subsequent 89% suppression of IAPP release was observed in the 4-h period following antisense treatment (1.78 +/- 0.13 (MSO3) vs 0.19 +/- 0.14 (O3) pmol/10(6) cells per 240 min, n = 7, P < 0.01). A significant increase in insulin mRNA (100 +/- 10% (MSO3) vs 124 +/- 8% (O3), n = 3, P < 0.05) and insulin content (13.0 +/- 0.9 (MSO3) vs 17.4 +/- 1.4 (O3) pmol/10(6) cells, n = 7, P = 0.028) was observed following treatment with O3 at 7.5 microM. O8, a 20-mer D-oligo directed to a region of IAPP mRNA further downstream than O3, also showed a decrease in IAPP mRNA and peptide release and an increase in insulin content. No significant changes were observed in the expression and release of the unrelated beta-cell peptide, neuropeptide Y. We thus show a suppression of synthesis and release of IAPP in HIT-T15 cells using antisense oligos. The associated increase in insulin mRNA and content in these cells after treatment with IAPP antisense oligos is in accord with an inhibitor action of IAPP on insulin availability.     

The structure-selectivity and sequence-preference of the junction-resolving enzyme CCE1 of Saccharomyces cerevisiae

CCE1 is a DNA junction-resolving enzyme involved in the resolution of recombining mitochondrial DNA in Saccharomyces cerevisiae. The CCE1 gene was cloned by PCR, and the expressed protein purified to homogeneity. CCE1 was found to bind to four-way DNA junctions, with a strong structural selectivity. The enzyme binds DNA junctions as a dimer, with slow subunit exchange occurring in free solution. While CCE1 binds equally to synthetic four-way DNA junctions of any sequence, it exhibits pronounced sequence-selectivity in cleavage. Both fixed junctions and those capable of branch migration can be cleaved, with a preference for cleavage at the sequence 5'CT/. Cleavage of junctions tethered to adopt specific stacking isomers demonstrated that the target sequences are cleaved fivefold faster when located on a continuous strand compared to an exchanging strand.     

Expression and regulation of constitutive and acute phase serum amyloid A mRNAs in hepatic and non-hepatic cell lines

A 60 kb conjugative plasmid, pND300, which encodes nisin resistance, was identified in Lactococcus lactis ssp. lactis (L. lactis) M189. pND300 was found to mobilize the transfer of some other plasmids as indicated by the mobilization of plasmids encoding lactose utilization. The nisin resistance determinant from pND300 was initially subcloned on a 12 kb DNA fragment and subsequently reduced to 10.4 kb. Restriction analysis, PCR, Southern hybridization and sequencing illustrated that the nisin resistance of pND300 is very similar to that encoded by the transposon involved in nisin production. pND300 encodes nisR as well as nisK and the recently reported nisF, nisE and nisG, but does not encode nisI. The DNA fragment encoding the nis genes is flanked by IS946 with a copy at each end in reverse orientation. The expression of these nis genes is probably controlled by a putative promoter upstream of nisR, which is composed of the TTGCAA hexanucleotide on the insertion sequence IS946 and the TATAAT sequence 21 bp downstream.     

Pathways of glutathione metabolism and transport in isolated proximal tubular cells from rat kidney

Cellular uptake and metabolism of exogenous glutathione (GSH) in freshly isolated proximal tubular (PT) cells from rat kidney were examined in the absence and presence of inhibitors of GSH turnover [acivicin, L-buthionine-S,R-sulfoximine (BSO)] to quantify and assess the role of different pathways in the handling of GSH in this renal cell population. Incubation of PT cells with 2 or 5 mM GSH in the presence of acivicin/BSO produced 3- to 4-fold increases in intracellular GSH within 10-15 min. These significantly higher intracellular concentrations were maintained for up to 60 min. At lower concentrations of extracellular GSH, an initial increase in intracellular GSH concentrations was observed, but this was not maintained for the 60-min time course. In the absence of inhibitors, intracellular concentrations of GSH increased to levels that were 2- to 3-fold higher than initial values in the first 10-15 min, but these dropped below initial levels thereafter. In both the absence and presence of acivicin/BSO, PT cells catalyzed oxidation of GSH to glutathione disulfide (GSSG) and degradation of GSH to glutamate and cyst(e)ine. Exogenous tert-butyl hydroperoxide oxidized intracellular GSH to GSSG in a concentration-dependent manner and extracellular GSSG was transported into PT cells, but limited intracellular reduction of GSSG to GSH occurred. Furthermore, incubation of cells with precursor amino acids produced little intracellular synthesis of GSH, suggesting that PT cells have limited biosynthetic capacity for GSH under these conditions. Hence, direct uptake of GSH, rather than reduction of GSSG or resynthesis from precursors, may be the primary mechanism to maintain intracellular thiol redox status under toxicological conditions. Since PT cells are a primary target for toxicants, the ability of these cells to rapidly take up and metabolize GSH may serve as a defensive mechanism to protect against chemical injury.     

Structural determinants of the catalytic reactivity of the buried cysteine of Escherichia coli thioredoxin

The structurally homologous thioredoxins and thioltransferases/glutaredoxins possess a solvent-exposed cysteine sulfur which carries out a nucleophilic attack on the target disulfide as well as a structurally adjacent solvent inaccessible thiol. The mechanistic basis of the essentially exclusive redox reactivity of the thioredoxins in contrast to the thiol-disulfide exchange reactions characteristic of the thioltransferases lies in the relative reactivity of the buried cysteine. A stable analog of the mixed disulfide state of Escherichia coli thioredoxin is used to demonstrate a pK value of 11.1 for the solvent inaccessible Cys 35 thiol. NMR chemical shift pH titration analysis indicates a very low dielectric surrounding the Cys 35 sulfur providing a basis for both the elevated pK and the enhanced apparent nucleophilicity. The buried Asp 26 likely serves as the proton sink for the (de)protonation of Cys 35. Relevance to the reactivity of the mammalian protein isomerases is discussed.     

Intraclass correlation among measures related to alcohol use by school aged adolescents: estimates, correlates and applications in intervention studies

School-based alcohol use prevention studies frequently employ designs in which schools are assigned to treatment conditions while observations are made on individuals. The nesting of schools within treatment conditions requires that the treatment effect be assessed against the between-school variance; unfortunately, that variance is usually larger than for randomly constituted groups and its precision is usually less than that for the within-school variance. These factors often combine to reduce power substantially. To address these problems, investigators need good estimates for the intraclass correlation which together with the number of observations per school determines the magnitude of the extra variation in the nested design. This article presents estimates of school-level intraclass correlation for measures related to alcohol use among ninth and twelfth grade students and discusses their use in planning new studies and analyzing previous or current studies.     

Abnormal lignin in a loblolly pine mutant

Novel lignin is formed in a mutant loblolly pine (Pinus taeda L.) severely depleted in cinnamyl alcohol dehydrogenase (E.C. 1.1.1.195), which converts coniferaldehyde to coniferyl alcohol, the primary lignin precursor in pines. Dihydroconiferyl alcohol, a monomer not normally associated with the lignin biosynthetic pathway, is the major component of the mutant's lignin, accounting for approximately 30 percent (versus approximately 3 percent in normal pine) of the units. The level of aldehydes, including new 2-methoxybenzaldehydes, is also increased. The mutant pines grew normally indicating that, even within a species, extensive variations in lignin composition need not disrupt the essential functions of lignin.     

Intravitreal invasion of malignant cells from choroidal melanoma after brachytherapy

We report intravitreal invasion by melanoma cells from a choroidal melanoma after brachytherapy. A malignant melanoma of the choroid with collar-button configuration was treated with iodine 125 brachytherapy. Years later, the collar button developed a dark-chocolate color and began shedding pigmented debris into the vitreous. Coalescence of this debris into spheroidal aggregates suggested the presence of malignant cells; the eye was enucleated. Histologic sections demonstrated a choroidal melanoma with intraretinal and intravitreal invasion by melanoma. Clinical evidence of intraretinal invasion by melanoma cells along with pigmented debris within the vitreous cavity, especially when clustered in spheroidal aggregates, suggests the presence of intravitreal invasion by malignant cells. In this case, intravitreal invasion was verified histologically.