Early expression of vimenti in human mammary cultures

Summary The intermediate filaments of most epithelial cells in vivo consist solely of cytokeratins. Using monoclonal antibodies to vimentin or keratin, we have examined the expression of vimentin in homologous specimens of frozen tissue sections and primary cultures of normal human mammary epithelium. In frozen sections, only epithelial cells reacted with the antikeratin antibody, whereas antivimentin reactivity was associated with stromal cells. All epithelial cultures were positive for cytokeratin and in addition coexpressed vimentin as strongly as cultured fibroblasts and as early as the 4th d after initiation of the culture. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis of cytoskeletal preparations of secondary cultures of normal mammary epithelium have also demonstrated the appearance of a moiety identical to the vimentin found in cultured fibroblasts. Our observations are consistent with the hypothesis that vimentin expression is induced, possibly as a result of changes in cell shape or growth rate, when cells are freed from three-dimensional restirctions imposed by the tissue of origin     

Circadian resonance in the development of two sympatric species of Camponotus ants

Circadian clocks provide adaptive advantage to their owners by timing their behavioural and physiological processes in accordance with the external environment. Here we report the results of our study aimed at investigating the effect of the interaction between circadian timing system and environmental light/dark (LD) cycles on pre-adult development time of two sympatric species of Componotus ants, the night active Componotus compressus, and the day active C. paria—both species develop in dark underground nests, under fairly constant conditions of humidity and temperature. We estimated pre-adult developmental durations in these ants under three different LD cycles (T20 = 10 h of light and 10 h of darkness, T24 = 12 h of light and 12 h of darkness, and T28 = 14 h of light and 14 h of darkness). We find that both species exhibit significantly faster pre-adult development under T24 compared to T20 and T28. Given that faster development in insects is considered as an adaptive strategy these results can be taken to suggest that Camponotus ants accrue greater fitness advantage under T24 compared to T20 and T28 LD cycles, possibly due to “circadian resonance” between circadian timing system and environmental LD cycle. Thus our study reveals that boreal species of ants could serve as a case for the study of adaptive significance of circadian organization.     

The role of albumin and PPAR‐α in differentiation‐dependent change of fatty acid profile during differentiation of mesenchymal stem cells to hepatocyte‐like cells

Differentiation of mesenchymal stem cells (MSCs) to hepatocyte‐like cells is associated with morphological and biological changes. In this study, the effect of hepatogenic differentiation on fatty acid profile and the expression of proliferator‐activated receptors‐α (PPAR‐α) have been studied. For this purpose, MSCs isolated from human umbilical cord were differentiated into hepatocyte‐like cells on selective culture media. The morphological and biochemical changes, PPAR‐α expression and reactive oxygen species (ROS) levels were studied during the differentiation process. Besides, the cells were processed to determine changes in fatty acid profile using gas chromatography analysis. The results showed that hepatic differentiation of the MSCs is associated with a decrease in major polyunsaturated fatty acids in mature hepatocytes, whereas there was an increase in the saturated fatty acid (SFA) levels during hepatocyte maturation. The differentiation‐dependent shift in the ratio of SFA/USFA was associated with changes in albumin and PPAR‐α expression, whereas changes in fatty acid profile were independent of ROS production and lipid peroxidation in differentiating cells. In conclusion, these data may suggest that hepatocyte formation during the stem cell differentiation is associated with a shift in the fatty acid profile that is probably a normal phenomenon in hepatogenic differentiation of the MSCs. Copyright © 2014 John Wiley & Sons, Ltd.     

Exposure of insect midgut cells to Sambucus nigra L. agglutinins I and II causes cell death via caspase-dependent apoptosis

Sambucus nigra agglutinins I and II, further referred to as SNA-I and SNA-II, are two ricin-related lectins from elderberry. SNA-I is a chimeric lectin composed of an A-chain with enzymatic activity and a B-chain with carbohydrate-binding activity, and therefore belongs to the group of type 2 ribosome-inactivating proteins. In contrast, SNA-II consists only of carbohydrate-binding B-chains. The physiological effect of SNA-I was tested on different insect cell lines (midgut, ovary, fat body, embryo). In sensitive midgut CF-203 cells, SNA-I induced cell death with typical characteristics such as cell shrinkage, plasma membrane blebbing, nuclear condensation and DNA fragmentation. The effect was dose-dependent with 50% death of 4-day-exposed cells at 3 nM. SNA-I exposure induced caspase-3 like activities, suggesting that SNA-I can induce the apoptotic pathway. Interestingly, the hololectin SNA-II also induced apoptosis in CF-203 cells at similar doses with the same physiological events. SNA-I and SNA-II both induced caspase-dependent apoptosis at low concentrations (nM order), leading to typical symptoms of cell death in sensitive cells. This effect seems independent from the catalytic activity of the A-chain, but depends on the carbohydrate-binding B-chain.     

Mutation in NSUN2, which encodes an RNA methyltransferase, causes autosomal-recessive intellectual disability

Causes of autosomal-recessive intellectual disability (ID) have, until very recently, been under researched because of the high degree of genetic heterogeneity. However, now that genome-wide approaches can be applied to single multiplex consanguineous families, the identification of genes harboring disease-causing mutations by autozygosity mapping is expanding rapidly. Here, we have mapped a disease locus in a consanguineous Pakistani family affected by ID and distal myopathy. We genotyped family members on genome-wide SNP microarrays and used the data to determine a single 2.5 Mb homozygosity-by-descent (HBD) locus in region 5p15.32-p15.31; we identified the missense change c.2035G>A (p.Gly679Arg) at a conserved residue within NSUN2. This gene encodes a methyltransferase that catalyzes formation of 5-methylcytosine at C34 of tRNA-leu(CAA) and plays a role in spindle assembly during mitosis as well as chromosome segregation. In mouse brains, we show that NSUN2 localizes to the nucleolus of Purkinje cells in the cerebellum. The effects of the mutation were confirmed by the transfection of wild-type and mutant constructs into cells and subsequent immunohistochemistry. We show that mutation to arginine at this residue causes NSUN2 to fail to localize within the nucleolus. The ID combined with a unique profile of comorbid features presented here makes this an important genetic discovery, and the involvement of NSUN2 highlights the role of RNA methyltransferase in human neurocognitive development.     

A Clinical Tool for Reducing Central Nervous System Depression among Neonates Exposed to Codeine through Breast Milk

Background

Neonates are commonly exposed to maternal codeine through breast milk. Central Nervous System (CNS) depression has been reported in up to 24% of nurslings following codeine exposure. In 2009, we developed guidelines to improve the safety of codeine use during breastfeeding based on previously established pharmacogenetic and clinical risk factors. The primary objective of this study was to prospectively evaluate the effectiveness of these guidelines in ensuring neonatal safety.

Methods and Findings

Women taking codeine for pain following caesarean section were given safety guidelines, including advice to use the lowest codeine dose for no longer than four days and to switch to a non-opioid when possible. Mothers provided a saliva sample for analysis of genes involved in opioid disposition, metabolism and response. A total of 238 consenting women participated. Neonatal sedation was reported in 2.1% (5/238) of breastfeeding women taking codeine according to our safety guidelines. This rate was eight fold lower than that reported in previous prospective studies. Women reporting sedated infants were taking codeine for a significantly longer period of time (4.80±2.59 days vs. 2.52±1.58 days, p = 0.0018). While following the codeine safety guidelines, mothers were less likely to supplement with formula, reported lower rates of sedation in themselves and breastfed more frequently throughout the day when compared to previously reported rates. Genotyping analysis of cytochrome p450 2D6 (CYP2D6), uridine-diphosphate glucuronosyltransferase (UGT) 2B7, p-glycoprotein (ABCB1), the mu-opioid receptor (OPRM1) and catechol-o-demethyltransferase (COMT) did not predict codeine response in breastfeeding mother/infant pairs when following the safety guidelines.

Conclusions

The only cases of CNS depression occurred when the length of codeine use exceeded the guideline recommendations. Neonatal safety of codeine can be improved using evidence-based guidelines, even in those deemed by genetics to be at high risk for toxicity.     

A Rapid Method for Purification of Oligonucleotides

Abstract

A simple, rapid and novel method for purification of the oligonucleotide using silica gel matrix is described.