Electric Field-Controlled Benzoic Acid and Sulphanilamide Delivery from Poly(Vinyl Alcohol) Hydrogel

The controlled release of benzoic acid (3.31 Å) and sulphanilamide (3.47 Å) from poly(vinyl alcohol), PVA, hydrogels fabricated by solution casting at various cross-linking ratios, were investigated. The PVA hydrogels were characterized in terms of the degree of swelling, the molecular weight between cross-links, and the mesh size. The drug release experiment was carried out using a modified Franz diffusion cell, at a pH value of 5.5 and at temperature of 37°C. The amount of drug release and the diffusion coefficients of the drugs from the PVA hydrogels increased with decreasing cross-linking ratio, as a larger mesh size was obtained with lower cross-linking ratios. With the application of an electric field, the amount of drug release and the diffusion coefficient increased monotonically with increasing electric field strength, since the resultant electrostatic force drove the ionic drugs from the PVA matrix. The drug size, matrix pore size, electrode polarity, and applied electric field were shown to be influential controlling factors for the drug release rate.KEY WORDS: electrophoresis force, ionic drug delivery, iontophoresis, poly(vinyl alcohol)     

Deletion of the gene encoding the ubiquitously expressed glucose-6-phosphatase catalytic subunit-related protein (UGRP)/glucose-6-phosphatase catalytic subunit-beta results in lowered plasma cholesterol and elevated glucagon

In liver, glucose-6-phosphatase catalyzes the hydrolysis of glucose-6-phosphate (G6P) to glucose and inorganic phosphate, the final step in the gluconeogenic and glycogenolytic pathways. Mutations in the glucose-6-phosphatase catalytic subunit (G6Pase) give rise to glycogen storage disease (GSD) type 1a, which is characterized in part by hypoglycemia, growth retardation, hypertriglyceridemia, hypercholesterolemia, and hepatic glycogen accumulation. Recently, a novel G6Pase isoform was identified, designated UGRP/G6Pase-beta. The activity of UGRP relative to G6Pase in vitro is disputed, raising the question as to whether G6P is a physiologically important substrate for this protein. To address this issue we have characterized the phenotype of UGRP knock-out mice. G6P hydrolytic activity was decreased by approximately 50% in homogenates of UGRP(-/-) mouse brain relative to wild type tissue, consistent with the ability of UGRP to hydrolyze G6P. In addition, female, but not male, UGRP(-/-) mice exhibit growth retardation as do G6Pase(-/-) mice and patients with GSD type 1a. However, in contrast to G6Pase(-/-) mice and patients with GSD type 1a, UGRP(-/-) mice exhibit no change in hepatic glycogen content, blood glucose, or triglyceride levels. Although UGRP(-/-) mice are not hypoglycemic, female UGRP(-/-) mice have elevated ( approximately 60%) plasma glucagon and reduced ( approximately 20%) plasma cholesterol. We hypothesize that the hyperglucagonemia prevents hypoglycemia and that the hypocholesterolemia is secondary to the hyperglucagonemia. As such, the phenotype of UGRP(-/-) mice is mild, indicating that G6Pase is the major glucose-6-phosphatase of physiological importance for glucose homeostasis in vivo.     

Chromosome-Specific Painting in Cucumis Species Using Bulked Oligonucleotides

Chromosome-specific painting is a powerful technique in molecular cytogenetic and genome research. We developed an oligonucleotide (oligo)-based chromosome painting technique in cucumber (Cucumis sativus) that will be applicable in any plant species with a sequenced genome. Oligos specific to a single chromosome of cucumber were identified using a newly developed bioinformatic pipeline and then massively synthesized de novo in parallel. The synthesized oligos were amplified and labeled with biotin or digoxigenin for use in fluorescence in situ hybridization (FISH). We developed three different probes with each containing 23,000–27,000 oligos. These probes spanned 8.3–17 Mb of DNA on targeted cucumber chromosomes and had the densities of 1.5–3.2 oligos per kilobases. These probes produced FISH signals on a single cucumber chromosome and were used to paint homeologous chromosomes in other Cucumis species diverged from cucumber for up to 12 million years. The bulked oligo probes allowed us to track a single chromosome in early stages during meiosis. We were able to precisely map the pairing between cucumber chromosome 7 and chromosome 1 of Cucumis hystrix in a F₁ hybrid. These two homeologous chromosomes paired in 71% of prophase I cells but only 25% of metaphase I cells, which may provide an explanation of the higher recombination rates compared to the chiasma frequencies between homeologous chromosomes reported in plant hybrids.     

High-throughput screening of mouse knockout lines identifies true lean and obese phenotypes

We developed a high-throughput approach to knockout (KO) and phenotype mouse orthologs of the 5,000 potential drug targets in the human genome. As part of the phenotypic screen, dual-energy X-ray absorptiometry (DXA) technology estimates body-fat stores in eight KO and four wild-type (WT) littermate chow-fed mice from each line. Normalized % body fat (nBF) (mean KO % body fat/mean WT littermate % body fat) values from the first 2322 lines with viable KO mice at 14 weeks of age showed a normal distribution. We chose to determine how well this screen identifies body-fat phenotypes by selecting 13 of these 2322 KO lines to serve as benchmarks based on their published lean or obese phenotype on a chow diet. The nBF values for the eight benchmark KO lines with a lean phenotype were > or =1 s.d. below the mean for seven (perilipin, SCD1, CB1, MCH1R, PTP1B, GPAT1, PIP5K2B) but close to the mean for NPY Y4R. The nBF values for the five benchmark KO lines with an obese phenotype were >2 s.d. above the mean for four (MC4R, MC3R, BRS3, translin) but close to the mean for 5HT2cR. This screen also identifies novel body-fat phenotypes as exemplified by the obese kinase suppressor of ras 2 (KSR2) KO mice. These body-fat phenotypes were confirmed upon studying additional cohorts of mice for KSR2 and all 13 benchmark KO lines. This simple and cost-effective screen appears capable of identifying genes with a role in regulating mammalian body fat.     

Aphidicolin-Induced FRA3B Breakpoints Cluster in Two Distinct Regions

The common fragile site at chromosomal band 3p14.2 (FRA3B) is the most sensitive single site in the human genome to induced chromosomal lesions. This fragile site may predispose chromosome 3p to breakage that is commonly observed in lung, renal, and many other cancers. We previously used aphidicolin induction of FRA3B expression in a chromosome 3-only somatic cell hybrid to generate a series of hybrids with breakpoints in the 3p14.2 region. These breakpoints were localized to two distinct clusters, separated by 200 kb, that lie on either side of a region of frequent breakage within FRA3B as observed by FISH analysis. Seven proximal aphidicolin-induced breakpoints were localized at or near the end of a THE element. The THE-1 element is flanked by LINE andAlurepetitive elements. The eight distal aphidicolin-induced breakpoints clustered in a region capable of forming multiple hairpin-like structures. Thus repetitive elements and hairpin-like structures may be responsible for chromosome fragility in this region.     

Analgesic,anti-inflammatory and anti-ulcer properties of Thai Perilla frutescence fruit oil in animals

Perilla frutescens fruit oil (PFO) is rich in α-linolenic acid (ALA) and exhibits biological activities. We aimed to investigate analgesic, anti-inflammatory and anti-ulcer activities of PFO and PFO-supplemented soybean milk (PFO-SM) in animal models. Analgesic activity was assessed in acetic acid-induced writhing in mice, while anti-inflammatory activity was performed in ethyl phenylpropiolate (EPP)-induced ear edema and carrageenan-induced hind paw edema in rats. Anti-ulcer effects were conducted in water immersion stress, HCl/ethanol and indomethacin-induced gastric ulcer in rats. Distinctly, PFO, containing 6.96 mg ALA and 2.61 mg LA equivalence/g, did not induce acute toxicity (LD₅₀ > 10 mL/kg) in mice. PFO (2.5 and 5 mL/kg) and PFO-SM (0.05 mL PFO equivalence/kg) inhibited incidences of writhing (16.8, 18.0 and 32.3%, respectively) in acetic acid-induced mice. In addition, topical applications of PFO (0.1 and 1 mL/ear) significantly inhibited EPP-induced ear edema (59.3 and 65.7%, respectively) in rats, while PFO-SM slightly inhibited ear edema (25.9%). However, PFO and PFO-SM did not inhibit carrageenan-induced hind paw edema in rats. Indeed, PFO (2.5 and 5 mL/kg) significantly inhibited gastric ulcers in rats that induced by water immersion stress (92.4 and 96.6%, respectively), HCl/ethanol (74.8 and 73.3%, respectively) and indomethacin (68.8 and 88.9%, respectively), while PFO-SM did not. PFO displayed potent analgesic, anti-inflammatory and anti-ulcer properties, while PFO-SM exerted only analgesic properties. Thus, Thai PFO and its functional drink offer potential benefits in treatment of analgesic, inflammatory diseases and gastric ulcer.