Randomized Clinical Trial of a Self-Adhering Flowable Composite for Class I Restorations: 2-Year Results.

Objectives. To compare the clinical performances of a self-adhering resin composite and a conventional flowable composite with a self-etch bonding system on permanent molars. The influence of using rubber dam versus cotton roll isolation was also investigated. Materials and Methods. Patients aged between 6 and 12 years and presenting at least two permanent molars in need of small class I restorations were selected. Thirty-four pairs of restorations were randomly placed by the same operator. Fifteen patients were treated under rubber dam and nineteen using cotton rolls isolation and saliva ejector. They were evaluated according to the modified USPHS criteria at baseline, 6 months, and 1 and 2 years by two independent evaluators. Results. All patients attended the two-year recall. For all measured variables, there was no significant difference between rubber dam and cotton after 2 years of restoration with Premise Flowable or Vertise Flow (p value > 0.05). The percentage of restorations scored alpha decreased significantly over time with Premise Flowable and Vertise Flow for marginal adaptation and surface texture as well as marginal discoloration while it did not vary significantly for color matching. After 2 years, Vertise Flow showed a similar behaviour to the Premise Flowable used with a self-adhesive resin system.

Larvicidal activity of essential oils extracted from commonly used herbs in Lebanon against the seaside mosquito, Ochlerotatus caspius.

This study investigates the potential of essential oils from commonly used medical and culinary herbs in Lebanon as an environmentally safe measure to control the seaside mosquito, Ochlerotatus caspius. The composition of essential oils extracted from parsley seeds and leaves, alpine thyme inflorescences, anis seeds, and coriander fruits were analyzed by GC-MS, and the major components of these oils were found to be thymol, sabinene, carvacrol, anethole, and linalool, respectively. Mosquito larvicidal assays were conducted to evaluate the LC(50) and LC(90) after 24 and 48h of the essential oils and their major constituents. All of the tested oils proved to have strong larvicidal activity (LC(50): 15-156ppm) against Oc. caspius fourth instars, with the most potent oil being thyme inflorescence extract, followed by parsley seed oil, aniseed oil, and then coriander fruit oil. Toxicity of each oil major constituent was also estimated and compared to a reported larvicidal compound, eugenol.

Evidence for a role of Sky1p-mediated phosphorylation in 3' splice site recognition involving both Prp8 and Prp17/Slu4.

The SRPK family of kinases is specific for RS domain-containing splicing factors and known to play a critical role in protein-protein interaction and intracellular distribution of their substrates in both yeast and mammalian cells. However, the function of these kinases in pre-mRNA splicing remains unclear. Here we report that SKY1, a SRPK family member in Saccharomyces cerevisiae, genetically interacts with PRP8 and PRP17/SLU4, both of which are involved in splice site selection during pre-mRNA splicing. Prp8 is essential for splicing and is known to interact with both 5' and 3' splice sites in the spliceosomal catalytic center, whereas Prp17/Slu4 is nonessential and is required only for efficient recognition of the 3' splice site. Interestingly, deletion of SKY1 was synthetically lethal with all prp17 mutants tested, but only with specific prp8 alleles in a domain implicated in governing fidelity of 3'AG recognition. Indeed, deletion of SKY1 specifically suppressed 3'AG mutations in ACT1-CUP1 splicing reporters. These results suggest for the first time that 3' AG recognition may be subject to phosphorylation regulation by Sky1p during pre-mRNA splicing.

Comparison of gas chromatography and immunoassay methods in measuring the distribution of dieldrin in rainbow trout tissues.

Studies have been conducted to determine the distribution of dieldrin in various tissues of rainbow trout when exposed to several dieldrin concentrations. Medium sized fish with an average weight and length of 195.4 +/- 30.5 g and 25.7 +/- 1.4 cm, respectively, were placed in groups of 6 in 300 L tanks containing purified and aerated water and maintained at 10 degrees C. Following an acclimatization period of 10 days, each group of fish was exposed to one of four dieldrin concentrations ranging from 50 to 80 ppb. After 24 hours, the fish were taken out of the tanks and sacrificed. The brain, gills, liver, muscles and skin were collected from each fish. Dieldrin was extracted from each tissue using SPE techniques and analyzed by both gas chromatography (GC) and enzyme linked immunosorbent assay (ELISA). Results of analyses by the two techniques were highly correlated. The results also showed that liver and skin tissues had the highest level of dieldrin residues. In comparing the means of the six fish samples, it was found that liver or skin contained about 1.5-fold the level in brain, about 4.0 fold the level in muscles and about 6.5 fold the level in gills. Immunoassay proved to be as reliable an analytical tool as gas chromatography in this case.

The fate and persistence of zineb, maneb, and ethylenethiourea on fresh and processed tomatoes.

Tomatoes grown under greenhouse conditions were sprayed with radiolabelled maneb and zineb to determine the extent of degradation of these fungicides to ethylenethiourea (ETU) and to study the persistence of ETU on the fruits. The total (14C) residues decreased from 0.082 mg/kg and 0.11 mg/kg at day 0 to 0.023 mg/kg and 0.05 mg/kg at day 20, on zineb- and maneb-treated fruits, respectively. This reduction was mainly due to the rapid growth of the fruits. ETU residues on tomato fruits were found to decline with time. A sharp reduction in ETU content was observed during the first 24 h after treatment, followed by a slow decline in the following 5 days. ETU content was reduced by about 80% by day 20 after the fungicide application, and the concentration of EU, the major degradation product of ETU, doubled during the same period. Studies with tomatoes fortified with (14C) ETU (0.006 mg/kg) prior to processing into tomato paste showed that 70% of the radioactivity was lost during washing of the tomatoes in water. Further losses of ETU occurred during boiling of the juice (6%) and during storage of the tomato paste for a period of 3 weeks (3%).

Comparison of gas chromatography and immunoassay methods in quantifying fenitrothion residues in grape juice processed into alcoholic drinks.

Wine and Arak, the national alcoholic drink in Lebanon, were prepared from grape juice fortified with fenitrothion to a concentration of 20ppm. Samples of the 11 fractions produced by the fermentation and distillation steps were analyzed for fenitrothion residues using gas chromatography (GC) and enzyme-linked immunosorbent assay (ELISA). Results of residue analyses showed that the two techniques were highly correlated (r = 0.978) and indicated that fenitrothion was stable during the fermentation steps but not during distillation. The clarified wine 35 days later contained about 85% (15.3 ppm) of the fenitrothion concentration found in the juice as determined by GC analysis. Arak was prepared by a two-steps distillation of the clarified wine. The alcohol distillate and undistilled fraction from the first distillation contained 2.5 ppm and 5.8 ppm of fenitrothion, respectively. No fenitrothion residues were detected by both techniques in the four fractions collected from the second distillation step.

Relationship of dietary intake to DDE residues in breast milk of nursing mothers in Beirut.

Milk samples were collected from 32 nursing mothers living in the Beirut area, Lebanon. Dietary intakes of participating mothers were obtained from data of their diet histories, 24 h dietary recalls and food frequency questionnaires. Milk samples were screened for the presence of organochlorine pesticide residues and DDE levels were estimated using gas chromatographic techniques. The relationship between consumption of various food groups and DDE content of milk was investigated. A positive correlation was found between the consumption of either/or high fat meat, tuna fish and DDE levels in milk. Consumption of poultry products showed a weak correlation with DDE content of milk, whereas consumption of vegetable oils showed a negative correlation.

Functional characterization of a Nup159p-containing nuclear pore subcomplex.

Nup159p/Rat7p is an essential FG repeat-containing nucleoporin localized at the cytoplasmic face of the nuclear pore complex (NPC) and involved in poly(A)+ RNA export and NPC distribution. A detailed structural-functional analysis of this nucleoporin previously demonstrated that Nup159p is anchored within the NPC through its essential carboxyl-terminal domain. In this study, we demonstrate that Nup159p specifically interacts through this domain with both Nsp1p and Nup82p. Further analysis of the interactions within the Nup159p/Nsp1p/Nup82p subcomplex using the nup82Delta108 mutant strain revealed that a deletion within the carboxyl-terminal domain of Nup82p prevents its interaction with Nsp1p but does not affect the interaction between Nup159p and Nsp1p. Moreover, immunofluorescence analysis demonstrated that Nup159p is delocalized from the NPC in nup82Delta108 cells grown at 37 degrees C, a temperature at which the Nup82Delta108p mutant protein becomes degraded. This suggests that Nup82p may act as a docking site for a core complex composed of the repeat-containing nucleoporins Nup159p and Nsp1p. In vivo transport assays further revealed that nup82Delta108 and nup159-1/rat7-1 mutant strains have little if any defect in nuclear protein import and protein export. Together our data suggest that the poly(A)+ RNA export defect previously observed in nup82 mutant cells might be due to the loss from the NPCs of the repeat-containing nucleoporin Nup159p.

Evidence for a role for galectin-1 in pre-mRNA splicing.

Galectins are a family of beta-galactoside-binding proteins that contain characteristic amino acid sequences in the carbohydrate recognition domain (CRD) of the polypeptide. The polypeptide of galectin-1 contains a single domain, the CRD. The polypeptide of galectin-3 has two domains, a carboxyl-terminal CRD fused onto a proline- and glycine-rich amino-terminal domain. In previous studies, we showed that galectin-3 is a required factor in the splicing of nuclear pre-mRNA, assayed in a cell-free system. We now document that (i) nuclear extracts derived from HeLa cells contain both galectins-1 and -3; (ii) depletion of both galectins from the nuclear extract either by lactose affinity adsorption or by double-antibody adsorption results in a concomitant loss of splicing activity; (iii) depletion of either galectin-1 or galectin-3 by specific antibody adsorption fails to remove all of the splicing activity, and the residual splicing activity is still saccharide inhibitable; (iv) either galectin-1 or galectin-3 alone is sufficient to reconstitute, at least partially, the splicing activity of nuclear extracts depleted of both galectins; and (v) although the carbohydrate recognition domain of galectin-3 (or galectin-1) is sufficient to restore splicing activity to a galectin-depleted nuclear extract, the concentration required for reconstitution is greater than that of the full-length galectin-3 polypeptide. Consistent with these functional results, double-immunofluorescence analyses show that within the nucleus, galectin-3 colocalizes with the speckled structures observed with splicing factor SC35. Similar results are also obtained with galectin-1, although in this case, there are areas of galectin-1 devoid of SC35 and vice versa. Thus, nuclear galectins exhibit functional redundancy in their splicing activity and partition, at least partially, in the nucleoplasm with another known splicing factor.

A structure/function analysis of Rat7p/Nup159p, an essential nucleoporin of Saccharomyces cerevisiae.

Rat7p/Nup159p is an essential nucleoporin of Sac-charomyces cerevisiae originally isolated in a genetic screen designed to identify yeast temperature-sensitive mutants defective in mRNA export. Here we describe a detailed structural-functional analysis of Rat7p/Nup159p. The mutation in the rat7-1 ts allele, isolated in the original genetic screen, was found to be a single base pair change that created a stop codon approximately 100 amino acids upstream of the actual stop codon of this 1,460 amino acid polypeptide, thus eliminating one of the two predicted coiled-coil regions located near the carboxyl terminus of the protein. These coiled-coil regions are essential since an allele lacking both coiled-coil regions was unable to support growth under any conditions. In contrast, no other region of the protein was absolutely required. The SAFG/PSFG repeat region in the central third of the protein was completely dispensable for growth at temperatures between 16 degrees C and 37 degrees C and cells expressing this mutant allele were indistinguishable from wild type. Deletion of the amino-terminal third of the protein, upstream from the repeat region, or the portion between the repeat region and the coiled-coils resulted in temperature-sensitivity, but the two alleles showed distinct phenotypes with respect to the behavior of nuclear pore complexes (NPCs). Taken together, our data suggest that Rat7p/Nup159p is anchored within the NPC through its coiled-coil region and adjacent sequences. In addition, we postulate that the N-terminal third of Rat7p/Nup159p plays an important role in mRNA export.

Identification of galectin-3 as a factor in pre-mRNA splicing.

Galectin-3 (M(r) approximately 35,000) is a galactose/lactose-specific lectin found in association with ribonucleoprotein complexes in many animal cells. Cell-free-splicing assays have been carried out to study the requirement for galectin-3 in RNA processing by HeLa cell nuclear extracts by using 32P-labeled MINX as the pre-mRNA substrate. Addition of saccharides that bind galectin-3 with high affinity inhibited product formation in the splicing assay, while addition of carbohydrates that do not bind to the lectin did not inhibit product formation. Nuclear extracts depleted of galectin-3 by affinity adsorption on a lactose-agarose column were deficient in splicing activity. Extracts subjected to parallel adsorption on control cellobiose-agarose retained splicing activity. The activity of the galectin-3-depleted extract could be reconstituted by the addition of purified recombinant galectin-3, whereas the addition of other lectins, either with a similar saccharide binding specificity (soybean agglutinin) or with a different specificity (wheat germ agglutinin), did not restore splicing activity. The formation of splicing complexes was also sensitive to galectin-3 depletion and reconstitution. Together, these results define a requirement for galectin-3 in pre-mRNA splicing and identify it as a splicing factor.

Phenotypic conversion of TK-deficient cells following electroporation of functional TK enzyme.

The ability to phenotypically rescue a mutant (Rat-3, thymidine kinase-deficient) cell line by electroporation of functional TK enzyme has been investigated. Extracts of electroporated cells showed a 35-fold increase in TK enzyme levels under conditions where greater than 90% of the cells remained viable. The electroporated enzyme was intracellular, as demonstrated by the fact that cells were able to utilize exogenous [3H]thymidine for DNA synthesis. By in situ autoradiography, 82% of electroporated cells contained functional enzyme and incorporated [3H]thymidine into DNA. Thus, this technique can efficiently provide a missing metabolic function to cultured mammalian cells.

Effect of Pasteurization, Centrifugation and Additives on Quality of Concentrated Yogurt (Labneh).

Pasteurization of yogurt by heat at 60-80°C or by addition of 0.2 to 2% hydrogen peroxide were investigated. Heat-treatment caused a very pronounced reduction in the bacterial count of yogurt samples. Addition of hydrogen peroxide was not effective at the low levels studied. Higher concentrations of hydrogen peroxide that were effective in reducing the bacterial population persisted in the food for more than 4 weeks. Heat-pasteurization in the presence of hydrogen peroxide, was very efficient in destroying bacterial cells and hastened the disappearance of hydrogen peroxide residues from the food. "Labneh", which is usually prepared from yogurt by filtering out the whey through a cloth, had an inferior texture when prepared from pasteurized yogurt. Milder heat treatment in the presence of hydrogen peroxide and addition of potassium sorbate and thickeners, greatly improved the quality and the shelf-life of labneh as confirmed by sensory evaluation experiments. Further improvements were possible by preparing labneh using centrifugation at various speeds for a short time.

Fate of DDT and parathion in grapes processed into arak, an alcoholic beverage.

Arak, the national alcoholic drink in Lebanon, was prepared from grapes to which either DDT or parathion had been added. Samples of the nine fractions produced from the fermentation and distillation steps were analyzed for DDT and parathion and their respective metabolites. DDT degraded to DDD during the fermentation step resulting in a sharp decrease in DDT level. The two distillation steps contributed to a further decrease in the DDT level so that the final product contained less than 2% of the amount found in the fresh grape juice. Although the concentration of DDD increased sharply during fermentation, it also decreased to a negligible level during the subsequent distillation procedure. Parathion was more stable than DDT during the fermentation and first distillation steps. However, the second distillation process caused a sharp decline in its level and the Arak contained only about 6% of the residues present in the fresh juice, paranithophenol being the only metabolite detected.

Association of glyceraldehyde-3-phosphate dehydrogenase with the particulate fraction of chicken skeletal muscle.

When chicken breast muscle was homogenized in water, approximately 86% of the glyceraldehyde-3-phosphate dehydrogenase was associated with the particulate fraction. The enzyme was solubilized by increasing pH with a very marked increase in the pH range of 6.9 to 7.1. At low ionic strength (about 0.015), approximately 50% of the enzyme is solubilized at pH 7.5 and above. Increasing ionic strength also led to increased solubilization. In addition, there was a specific cation effect with Ca2+ greater than Mg2+ greater than K+ greater than Na+ at a constant ionic strength. Glyceraldehyde 3-phosphate and 2,3-bisphosphoglycerate were effective in partially solubilizing the enzyme. Solubilized glyceraldehyde-3-phosphate dehydrogenase can rebind to the particulate fraction of the homogenized muscle. The soluble form of the enzyme has a higher V and a higher Km (glyceraldehyde-3-phosphate) than the enzyme bound to the particulate fraction.