Acute and subacute toxicity studies of Aegle marmelos Corr., an Indian medicinal plant.

This study was designed to elucidate the toxicity of the widely used plant Aegle marmelos in rats. We have taken total alcoholic, total aqueous, whole aqueous and methanolic extracts isolated from the leaves of A. marmelos and studied their toxic effects. Acute, subacute and LD(50) values were determined in experimental rats. The dead animals were obtained from primary screening studies, LD(50) value determination experiments and acute studies subjected to postmortem studies. The external appearance of the dead animals, the appearance of the viscera, heart, lungs, stomach, intestine, liver, kidney, spleen and brain were carefully noted and any apparent and significant features or differences from the norm were recorded. Following the chronic administration of A. marmelos for 14 days, the vital organs such as heart, liver, kidney, testis, spleen and brain were carefully evaluated by histopathological studies and any apparent and significant changes or differences from the norm were studied. From the acute administration of A. marmelos, the LD(50) values were determined using graphical method. The hearts stopped in systolic stand-still in the acute experiments. There were no remarkable changes noticed in the histopathological studies after 50 mg/kg body wt of the extracts of A. marmelos when administered intraperitoneally for 14 days successively. Pathologically, neither gross abnormalities nor histopathological changes were observed. After calculation of LD(50) values using graphical methods, we found a broad therapeutic window and a high therapeutic index value for A. marmelos extracts. Intraperitoneal administration of the extracts of the leaves of A. marmelos at doses of 50, 70, 90 and 100 mg/kg body wt for 14 consecutive days to male and female Wistar rats did not induce any short-term toxicity. Collectively, these data demonstrate that the extracts of the leaves of A. marmelos have a high margin of drug safety.     

Enzymic formation of p-hydroxybenzoate from p-hydroxycinnamate

An enzyme that converts p-hydroxycinnamate into p-hydroxybenzoate was found in rat liver. It is localized in the mitochondria and requires ATP. The activity is lost when the mitochondria are stored frozen overnight. Addition of magnesium chloride, cytochrome c, GSH, coenzyme A or potassium cyanide did not have any effect on the activity. When the rats were fed with alpha-p-chlorophenoxyisobutyrate, the rate of formation of p-hydroxybenzoate increased twofold. The reaction has some similar properties to fatty acid oxidation, but appears to be different in many respects.     

Air trapping on chest CT is associated with worse ventilation distribution in infants with cystic fibrosis diagnosed following newborn screening

Background

In school-aged children with cystic fibrosis (CF) structural lung damage assessed using chest CT is associated with abnormal ventilation distribution. The primary objective of this analysis was to determine the relationships between ventilation distribution outcomes and the presence and extent of structural damage as assessed by chest CT in infants and young children with CF.

Methods

Data of infants and young children with CF diagnosed following newborn screening consecutively reviewed between August 2005 and December 2009 were analysed. Ventilation distribution (lung clearance index and the first and second moment ratios [LCI, M₁/M₀ and M₂/M₀, respectively]), chest CT and airway pathology from bronchoalveolar lavage were determined at diagnosis and then annually. The chest CT scans were evaluated for the presence or absence of bronchiectasis and air trapping.

Results

Matched lung function, chest CT and pathology outcomes were available in 49 infants (31 male) with bronchiectasis and air trapping present in 13 (27%) and 24 (49%) infants, respectively. The presence of bronchiectasis or air trapping was associated with increased M₂/M₀ but not LCI or M₁/M₀. There was a weak, but statistically significant association between the extent of air trapping and all ventilation distribution outcomes.

Conclusion

These findings suggest that in early CF lung disease there are weak associations between ventilation distribution and lung damage from chest CT. These finding are in contrast to those reported in older children. These findings suggest that assessments of LCI could not be used to replace a chest CT scan for the assessment of structural lung disease in the first two years of life. Further research in which both MBW and chest CT outcomes are obtained is required to assess the role of ventilation distribution in tracking the progression of lung damage in infants with CF.     

Elevated basal slippage mutation rates among the Canidae

The remarkable responsiveness of dog morphology to selection is a testament to the mutability of mammals. The genetic sources of this morphological variation are largely unknown, but some portion is due to tandem repeat length variation in genes involved in development. Previous analysis of tandem repeats in coding regions of developmental genes revealed fewer interruptions in repeat sequences in dogs than in the orthologous repeats in humans, as well as higher levels of polymorphism, but the fragmentary nature of the available dog genome sequence thwarted attempts to distinguish between locus-specific and genome-wide origins of this disparity. Using whole-genome analyses of the human and recently completed dog genomes, we show that dogs possess a genome-wide increase in the basal germ-line slippage mutation rate. Building on the approach that gave rise to the initial observation in dogs, we sequenced 55 coding repeat regions in 42 species representing 10 major carnivore clades and found that a genome-wide elevated slippage mutation rate is a derived character shared by diverse wild canids, distinguishing them from other Carnivora. A similarly heightened slippage profile was also detected in rodents, another taxon exhibiting high diversity and rapid evolvability. The correlation of enhanced slippage rates with major evolutionary radiations suggests that the possession of a "slippery" genome may bestow on some taxa greater potential for rapid evolutionary change.     

Serine and threonine phosphorylation of the low density lipoprotein receptor-related protein by protein kinase Calpha regulates endocytosis and association with adaptor molecules

The low density lipoprotein receptor-related protein (LRP) is a large receptor that participates in endocytosis, signaling pathways, and phagocytosis of necrotic cells. Mechanisms that direct LRP to function in these distinct pathways likely involve its association with distinct cytoplasmic adaptor proteins. We tested the hypothesis that the association of various adaptor proteins with the LRP cytoplasmic domain is modulated by its phosphorylation state. Phosphoamino acid analysis of metabolically labeled LRP revealed that this receptor is phosphorylated at serine, threonine, and tyrosine residues within its cytoplasmic domain, whereas inhibitor studies identified protein kinase Calpha (PKCalpha) as a kinase capable of phosphorylating LRP. Mutational analysis identified critical threonine and serine residues within the LRP cytoplasmic domain that are necessary for phosphorylation mediated by PKCalpha. Mutating these threonine and serine residues to alanines generated a receptor that was not phosphorylated and that was internalized more rapidly than wild-type LRP, revealing that phosphorylation reduces the association of LRP with adaptor molecules of the endocytic machinery. In contrast, serine and threonine phosphorylation was necessary for the interaction of LRP with Shc, an adaptor protein that participates in signaling events. Furthermore, serine and threonine phosphorylation increased the interaction of LRP with other adaptor proteins such as Dab-1 and CED-6/GULP. These results indicate that phosphorylation of LRP by PKCalpha modulates the endocytic and signaling function of LRP by modifying its association with adaptor proteins.     

INHIBITION OF THE BIOSYNTHESIS OF ISOPRENOID COMPOUNDS BY PHENOLIC ACIDS IN THE RAT BRAIN

—The incorporation of [2-¹⁴C]mevalonate into nonsaponifiable lipids by rat brain homogenates is inhibited by phenolic acids derived from tyrosine. The phenyl acids derived from phenylalanine are inhibitory only at very high concentrations compared with phenolic acids. The brain is more sensitive to inhibition by the phenolic acids than the liver. These studies indicate a possible role for phenolic acids in the impairment of cerebral sterol metabolism in phenylketonuria.     

Inhibition of the biosynthesis of sterols by phenyl and phenolic compounds in rat liver

The presence of mitochondria increased the incorporation of [2-(14)C]mevalonate into sterols in a cell-free system from rat liver. Various phenyl and phenolic compounds inhibited the incorporation of mevalonate when added in vitro. p-Hydroxycinnamate, a metabolite of tyrosine, was the most powerful inhibitor among the compounds tested. Catechol, resorcinol and quinol were inhibitory at high concentrations. Organic acids lacking an aromatic ring were not inhibitory. Two hypocholesterolaemic drugs, Clofibrate (alpha-p-chlorophenoxyisobutyrate) and Clofenapate [alpha,4-(p-chlorophenyl)phenoxyisobutyrate], which are known to affect some step before the formation of mevalonate in the biosynthesis of cholesterol in vivo, showed inhibition at a step beyond the formation of mevalonate in vitro. The presence of the aromatic ring and the carboxyl group in a molecule appears to be necessary for the inhibition.     

The Role of ARF6 in Biliary Atresia

Background & Aims

Altered extrahepatic bile ducts, gut, and cardiovascular anomalies constitute the variable phenotype of biliary atresia (BA).

Methods

To identify potential susceptibility loci, Caucasian children, normal (controls) and with BA (cases) at two US centers were compared at >550000 SNP loci. Systems biology analysis was carried out on the data. In order to validate a key gene identified in the analysis, biliary morphogenesis was evaluated in 2-5-day post-fertilization zebrafish embryos after morpholino-antisense oligonucleotide knockdown of the candidate gene ADP ribosylation factor-6 (ARF6, Mo-arf6).

Results

Among 39 and 24 cases at centers 1 and 2, respectively, and 1907 controls, which clustered together on principal component analysis, the SNPs rs3126184 and rs10140366 in a 3’ flanking enhancer region for ARF6 demonstrated higher minor allele frequencies (MAF) in each cohort, and 63 combined cases, compared with controls (0.286 vs. 0.131, P = 5.94x10^-7, OR 2.66; 0.286 vs. 0.13, P = 5.57x10^-7, OR 2.66). Significance was enhanced in 77 total cases, which included 14 additional BA genotyped at rs3126184 only (p = 1.58x10^-2, OR = 2.66). Pathway analysis of the 1000 top-ranked SNPs in CHP cases revealed enrichment of genes for EGF regulators (p<1 x10^-7), ERK/MAPK and CREB canonical pathways (p<1 x10^-34), and functional networks for cellular development and proliferation (p<1 x10^-45), further supporting the role of EGFR-ARF6 signaling in BA. In zebrafish embryos, Mo-arf6 injection resulted in a sparse intrahepatic biliary network, several biliary epithelial cell defects, and poor bile excretion to the gall bladder compared with uninjected embryos. Biliary defects were reproduced with the EGFR-blocker AG1478 alone or with Mo-arf6 at lower doses of each agent and rescued with arf6 mRNA.

Conclusions

The BA-associated SNPs identify a chromosome 14q21.3 susceptibility locus encompassing the ARF6 gene. arf6 knockdown in zebrafish implicates early biliary dysgenesis as a basis for BA, and also suggests a role for EGFR signaling in BA pathogenesis.     

An overview of enzymatic production of biodiesel

Biodiesel production has received considerable attention in the recent past as a biodegradable and nonpolluting fuel. The production of biodiesel by transesterification process employing alkali catalyst has been industrially accepted for its high conversion and reaction rates. Recently, enzymatic transesterification has attracted much attention for biodiesel production as it produces high purity product and enables easy separation from the byproduct, glycerol. But the cost of enzyme remains a barrier for its industrial implementation. In order to increase the cost effectiveness of the process, the enzyme (both intracellular and extracellular) is reused by immobilizing in a suitable biomass support particle and that has resulted in considerable increase in efficiency. But the activity of immobilized enzyme is inhibited by methanol and glycerol which are present in the reacting mixture. The use of tert-butanol as solvent, continuous removal of glycerol, stepwise addition of methanol are found to reduce the inhibitory effects thereby increasing the cost effectiveness of the process. The present review analyzes these methods reported in literature and also suggests a suitable method for commercialization of the enzymatic process.