Liver fatty acid binding protein gene ablation potentiates hepatic cholesterol accumulation in cholesterol-fed female mice

Although liver fatty acid binding protein (L-FABP) is postulated to influence cholesterol homeostasis, the physiological significance of this hypothesis remains to be resolved. This issue was addressed by examining the response of young (7 wk) female mice to L-FABP gene ablation and a cholesterol-rich diet. In control-fed mice, L-FABP gene ablation alone induced hepatic cholesterol accumulation (2.6-fold), increased bile acid levels, and increased body weight gain (primarily as fat tissue mass). In cholesterol-fed mice, L-FABP gene ablation further enhanced the hepatic accumulation of cholesterol (especially cholesterol ester, 12-fold) and potentiated the effects of dietary cholesterol on increased body weight gain, again mainly as fat tissue mass. However, in contrast to the effects of L-FABP gene ablation in control-fed mice, biliary levels of bile acids (as well as cholesterol and phospholipids) were reduced. These phenotypic alterations were not associated with differences in food intake. In conclusion, it was shown for the first time that L-FABP altered cholesterol metabolism and the response of female mice to dietary cholesterol. While the biliary and lipid phenotype of female wild-type L-FABP+/+ mice was sensitive to dietary cholesterol, L-FABP gene ablation dramatically enhanced many of the effects of dietary cholesterol to greatly induce hepatic cholesterol (primarily cholesterol ester) and triacylglycerol accumulation as well as to potentiate body weight gain (primarily as fat tissue mass). Taken together, these data support the hypothesis that L-FABP is involved in the physiological regulation of cholesterol metabolism, body weight gain, and obesity.     

Exercise-induced changes in cardiac gene expression and its relation to spatial maze performance

Cognitive performance is sensitive to both neural and non-neural changes induced by physical activity and inactivity. This study investigated whether access to physical activity outside a standard laboratory animal cage affected cognitive performance as measured by navigation of a spatial maze. It also examined gene expression in heart tissue for genes associated with cardiovascular function given recent reports of cognitive impairment associated with hyperlipidemia. Furthermore, we measured expression of neural-regulatory genes typically expressed in brain, but also found in cardiac tissue. Male Sprague-Dawley rats (n = 72) were separated into three groups having different access to physical activity: none outside a standard cage, twice-weekly physical activity, and every other day exercise on a running wheel. Compared with a sedentary group, spatial maze performance was enhanced in animals that had access to physical activity, either twice-weekly in a large box or every other day on a running wheel. Both the cardiovascular and neural-related genes expressed in the heart were distinguished by access to physical activity. Several genes that are associated with heart rate, cholesterol biosynthesis, blood pressure, and cell adhesion regulation, including GJA1, FDFT1, EDN1, and CD36, differed in animals based on access to physical activity. Neural-related genes expressed in cardiac tissue associated with neurite outgrowth, neuroplasticity, and neurogenesis including RTN4, HOMER2, ACTB, NCDN, KIF5B, and HMGB2, were expressed differently among the three groups. Significant shifts in ten cardiovascular and neural-related gene expressions in cardiac tissue were associated with physical activity and may have influenced learning and performance on a spatial maze.     

The use of (GTG)5 oligonucleotide as an RAPD primer to type Campylobacter concisus

AIM: DNA fingerprinting using (GTG)(5) oligonucleotide as a primer in a random amplified polymorphic DNA (RAPD) assay was assessed by typing isolates of Campylobacter concisus strains, collected over a period of 8 years. METHODS AND RESULTS: RAPD analysis using the (GTG)(5) oligonucleotide as a primer was used to type 100 isolates of C. concisus comprising mostly isolates from children with diarrhoea. Using this method, 86% of the isolates were found to be genotypically diverse. Of these heterogeneous isolates, 25 of the strains were also shown to be genetically distinct in a previous study using pulsed field gel electrophoresis. The remaining isolates (14) could be classified into five profile groups based on the DNA fingerprinting patterns. The assay successfully identified epidemiologically linked strains from the unrelated genetically diverse pool of strains. CONCLUSIONS: Laboratory RADP typing using the (GTG)(5) primer proved to be useful in distinguishing related strains of C. concisus from a large pool of unrelated strains of this organism. SIGNIFICANCE AND IMPACT OF THE STUDY: RAPD typing using (GTG)(5) is a simple method that could be used to investigate the epidemiology of C. concisus. The results suggest that homologous lineages of C. concisus may exist within an otherwise heterogeneous species complex. However, these data need to be confirmed using a more robust typing method.     

Cytoplasmic expression systems triggered by mRNA yield increased gene expression in post-mitotic neurons

Non-viral vectors are promising vehicles for gene therapy but delivery of plasmid DNA to post-mitotic cells is challenging as nuclear entry is particularly inefficient. We have developed and evaluated a hybrid mRNA/DNA system designed to bypass the nuclear barrier to transfection and facilitate cytoplasmic gene expression. This system, based on co-delivery of mRNA(A64) encoding for T7 RNA polymerase (T7 RNAP) with a T7-driven plasmid, produced between 10- and 2200-fold higher gene expression in primary dorsal root ganglion neuronal (DRGN) cultures isolated from Sprague–Dawley rats compared to a cytomegalovirus (CMV)-driven plasmid, and 30-fold greater expression than the enhanced T7-based autogene plasmid pR011. Cell-free assays and in vitro transfections highlighted the versatility of this system with small quantities of T7 RNAP mRNA required to mediate expression at levels that were significantly greater than with the T7-driven plasmid alone or supplemented with T7 RNAP protein. We have also characterized a number of parameters, such as mRNA structure, intracellular stability and persistence of each nucleic acid component that represent important factors in determining the transfection efficiency of this hybrid expression system. The results from this study demonstrate that co-delivery of mRNA is a promising strategy to yield increased expression with plasmid DNA, and represents an important step towards improving the capability of non-viral vectors to mediate efficient gene transfer in cell types, such as in DRGN, where the nuclear membrane is a significant barrier to transfection.     

DNA damage in leukocytes of workers occupationally exposed to 1-bromopropane

1-bromopropane (1-BP; n-propyl bromide) (CAS No. 106-94-5) is an alternative to ozone-depleting chlorofluorocarbons that has a variety of potential applications as a degreasing agent for metals and electronics, and as a solvent vehicle for spray adhesives. Its isomer, 2-brompropane (2-BP; isopropyl bromide) (CAS No. 75-26-3) impairs antioxidant cellular defenses, enhances lipid peroxidation, and causes DNA damage in vitro. The present study had two aims. The first was to assess DNA damage in human leukocytes exposed in vitro to 1- or 2-BP. DNA damage was also assessed in peripheral leukocytes from workers with occupational exposure to 1-BP. In the latter assessment, start-of- and end-of-work week blood and urine samples were collected from 41 and 22 workers at two facilities where 1-BP was used as a solvent for spray adhesives in foam cushion fabrication. Exposure to 1-BP was assessed from personal-breathing zone samples collected for 1-3 days up to 8h per day for calculation of 8h time weighted average (TWA) 1-BP concentrations. Bromide (Br) was measured in blood and urine as a biomarker of exposure. Overall, 1-BP TWA concentrations ranged from 0.2 to 271 parts per million (ppm) at facility A, and from 4 to 27 ppm at facility B. The highest exposures were to workers classified as sprayers. 1-BP TWA concentrations were statistically significantly correlated with blood and urine Br concentrations. The comet assay was used to estimate DNA damage. In vitro, 1- or 2-BP induced a statistically significant increase in DNA damage at 1mM. In 1-BP exposed workers, start-of- and end-of-workweek comet endpoints were stratified based on job classification. There were no significant differences in DNA damage in leukocytes between workers classified as sprayers (high 1-BP exposure) and those classified as non-sprayers (low 1-BP exposure). At the facility with the high exposures, comparison of end-of-week values with start-of-week values using paired analysis revealed non-sprayers had significantly increased comet tail moments, and sprayers had significantly increased comet tail moment dispersion coefficients. A multivariate analysis included combining the data sets from both facilities, log transformation of 1-BP exposure indices, and the use of multiple linear regression models for each combination of DNA damage and exposure indices including exposure quartiles. The covariates were gender, age, smoking status, facility, and glutathione S-transferase M1 and T1 (GSTM1, GSTT1) polymorphisms. In the regression models, start-of-week comet tail moment in leukocytes was significantly associated with serum Br quartiles. End-of-week comet tail moment was significantly associated with 1-BP TWA quartiles, and serum Br quartiles. Gender, facility, and GSTM1 had a significant effect in one or more models. Additional associations were not identified from assessment of dispersion coefficients. In vitro and in vivo results provide limited evidence that 1-BP exposure may pose a small risk for increasing DNA damage.     

Assessment of plants from the Brassicaceae family as genetic models for the study of nickel and zinc hyperaccumulation

We report on the second phase of a programme to select a relative of Arabidopsis thaliana for use in large-scale molecular genetic studies of nickel (Ni) and zinc (Zn) hyperaccumulation. We also report on the relatedness among Thlaspi caerulescens accessions and the utility of using O-acetyl-L-serine as a marker for Ni and Zn hyperaccumulation potential. Twenty-seven new accessions of metal-accumulating species collected in the Czech Republic, France, Greece, Italy, Slovenia and the USA during Spring-Summer 2002 were evaluated. The criteria established for selection were hyperaccumulation of metals (Ni and Zn); compact growth habit; reasonable time to flowering; production of > or = 1000 seeds per plant; self-fertility; compact diploid genome; high sequence similarity to A. thaliana; > or = 0.1% transformation efficiency with easy selection. We conclude that the best candidate identified in the first phase was the best candidate overall: T. caerulescens accession St Félix de Pallières.     

Medical electricity and madness in the 18th century: the legacies of Benjamin Franklin and Jan Ingenhousz

Benjamin Franklin had at least two accidents that resulted in electricity passing through his brain. In addition, he witnessed a patient's similar accident and performed an experiment that showed how humans could endure shocks to the head without serious ill effects, other than amnesia. Jan Ingenhousz, Franklin's Dutch-born medical correspondent better known for his discovery of photosynthesis, also had a serious accident that sent electricity though his head and, in a letter to Franklin, he described how he felt unusually elated the next day. During the 1780s, Franklin and Ingenhousz encouraged leading French and English electrical "operators" to try shocking the heads of melancholic and other deranged patients in their wards. Although they did not state that they were responding to Ingenhousz's and Franklin's suggestions, Birch, Aldini, and Gale soon did precisely what Ingenhousz and Franklin had suggested. These practitioners did not appear to induce convulsions in their mentally ill patients, but they still reported notable successes.