Mitochondrial membrane permeabilisation by amyloid aggregates and protection by polyphenols

Alzheimer's disease and Parkinson's disease are neurodegenerative disorders characterised by the misfolding of proteins into soluble prefibrillar aggregates. These aggregate complexes disrupt mitochondrial function, initiating a pathophysiological cascade leading to synaptic and neuronal degeneration. In order to explore the interaction of amyloid aggregates with mitochondrial membranes, we made use of two in vitro model systems, namely: (i) lipid vesicles with defined membrane compositions that mimic those of mitochondrial membranes, and (ii) respiring mitochondria isolated from neuronal SH-SY5Y cells. External application of soluble prefibrillar forms, but not monomers, of amyloid-beta (Aβ₄₂ peptide), wild-type α-synuclein (α-syn), mutant α-syn (A30P and A53T) and tau-441 proteins induced a robust permeabilisation of mitochondrial-like vesicles, and triggered cytochrome c release (CCR) from isolated mitochondrial organelles. Importantly, the effect on mitochondria was shown to be dependent upon cardiolipin, an anionic phospholipid unique to mitochondria and a well-known key player in mitochondrial apoptosis. Pharmacological modulators of mitochondrial ion channels failed to inhibit CCR. Thus, we propose a generic mechanism of thrilling mitochondria in which soluble amyloid aggregates have the intrinsic capacity to permeabilise mitochondrial membranes, without the need of any other protein. Finally, six small-molecule compounds and black tea extract were tested for their ability to inhibit permeation of mitochondrial membranes by Aβ₄₂, α-syn and tau aggregate complexes. We found that black tea extract and rosmarinic acid were the most potent mito-protectants, and may thus represent important drug leads to alleviate mitochondrial dysfunction in neurodegenerative diseases.     

Genomic organization and chromosomal localization of a new repeat MS7, specific for heterochromatin of sex chromosomes in Microtus species voles of the arvalis group

The tandemly arranged MS4 repeat with monomeric units of 4.1 kb is species-specifically distributed in heterochromatin of sex chromosomes of four common vole species of genus Microtus, group arvalis [1, 2]. In this work, we studied the genomic organization of the MS4 homolog in euchromatin of the X chromosome of M. arvalis. It has been shown by analyzing the phage genomic clones that one MS4 copy makes a part of a monomeric unit exceeding 8.5 kb that also includes a new MS7 repeat and, possibly, LINE fragments. MS7 is located together with MS4 in heterochromatin of common vole sex chromosomes, but in a substantially lesser amount. Probably, as a result of an evolutionary transition of an original repeat from euchromatin of the X chromosome to heterochromatin of the Y chromosome, MS4 underwent multiple amplification, and MS7 spread throughout heterochromatin, being surrounded by the MS4 tandem arrays.     

Profiling gene expression using onto-express

Gene expression profiles obtained through microarray or data mining analyses often exist as vast data strings. To interpret the biology of these genetic profiles, investigators must analyze this data in the context of other information such as the biological, biochemical, or molecular function of the translated proteins. This is particularly challenging for a human analyst because large quantities of less than relevant data often bury such information. To address this need we implemented an automated routine, called Onto-Express (http://vortex.cs.wayne.edu:8080), to systematically translate genetic fingerprints into functional profiles. Using strings of accession or cluster identification numbers, Onto-Express searches the public databases and returns tables that correlate expression profiles with the cytogenetic locations, biochemical and molecular functions, biological processes, cellular components, and cellular roles of the translated proteins. The profiles created by Onto-Express fundamentally increase the value of gene expression analyses by facilitating the translation of quantitative value sets to records that contain biological implications.     

Design and synthesis of a β-lactamase activated 5-fluorouracil prodrug

An efficient synthesis of a 5-fluorouracil-cephalosporin prodrug is described for use against colorectal and other cancers in antibody and gene-directed therapies. The compound shows stability in aqueous media until specifically activated by β-lactamase (βL). The kinetic parameters of the 5-fluorouracil-cephalosporin conjugate were determined in the presence of Enterobacter cloacae P99 βL (ECl βL) revealing a K_m = 95.4 μM and V_max = 3.21 μMol min^?1 mg^?1. The data compare favorably to related systems that have been reported and enable testing of this prodrug against cancer cell lines in vitro and in vivo.     

Fluoride Exposure Aggravates the Testicular Damage and Sperm Quality in Diabetic Mice: Protective Role of Ginseng and Banaba

The current study was conducted to investigate the effects of dietary zinc oxide (ZnO) on the antioxidant capacity, small intestine development, and jejunal gene expression in weaned piglets. Ninety-six 21-day-old piglets were randomly assigned to three dietary treatments. Each treatment had eight replicates with four piglets per replicate. The piglets were fed either control diet (control) or control diet supplemented with in-feed antibiotics (300 mg/kg chlortetracycline and 60 mg/kg colistin sulfate) or pharmacological doses of ZnO (3000 mg/kg). The experiment lasted 4 weeks. Blood samples were collected at days 14 and 28, while intestinal samples were harvested at day 28 of the experiment. Dietary high doses of ZnO supplementation significantly increased the body weight (BW) at day 14 and average daily gain (ADG) of days 1 to 14 in weaned piglets, when compared to control group (P < 0.05). The incidence of diarrhea of piglets fed ZnO-supplemented diets, at either days 1 to 14, days 14 to 28, or the overall experimental period, was significantly decreased in comparison with those in other groups (P < 0.05). Supplementation with ZnO increased the villus height of the duodenum and ileum in weaned piglets and decreased the crypt depth of the duodenum, when compared to the other groups (P < 0.05). Dietary ZnO supplementation decreased the malondialdehyde (MDA) concentration at either day 14 or day 28, but increased total superoxide dismutase (T-SOD) at day 14, when compared to that in the control (P < 0.05). ZnO supplementation upregulated the messenger RNA (mRNA) expression of zonula occludens-1 (ZO-1) and occludin in the jejunum mucosa of weaned piglets, compared to those in the control (P < 0.05). The pro-inflammatory cytokine interleukin-lβ (IL-1β) mRNA expression in the jejunum mucosa was downregulated in the ZnO-supplemented group, compared with the control (P < 0.05). Both in-feed antibiotics and ZnO supplementation decreased the mRNA expression of interferon-γ (IFN-γ), but increased the mRNA expression of transforming growth factor-β (TGF-β), in the jejunum mucosa of piglets, when compared to those in the control (P < 0.05). In summary, supplemental ZnO was effective on the prevention of post-weaning diarrhea (PWD) in weaned piglets and showed comparative growth-promoting effect on in-feed antibiotics, probably by the mechanism of improvement of the antioxidant capacity, restoration of intestinal barrier function and development, and modulation of immune functions.     

Molecular phylogenetics reveal multiple tertiary vicariance origins of the African rain forest trees

Background  

Tropical rain forests are the most diverse terrestrial ecosystems on the planet. How this diversity evolved remains largely unexplained. In Africa, rain forests are situated in two geographically isolated regions: the West-Central Guineo-Congolian region and the coastal and montane regions of East Africa. These regions have strong floristic affinities with each other, suggesting a former connection via an Eocene pan-African rain forest. High levels of endemism observed in both regions have been hypothesized to be the result of either 1) a single break-up followed by a long isolation or 2) multiple fragmentation and reconnection since the Oligocene. To test these hypotheses the evolutionary history of endemic taxa within a rain forest restricted African lineage of the plant family Annonaceae was studied. Molecular phylogenies and divergence dates were estimated using a Bayesian relaxed uncorrelated molecular clock assumption accounting for both calibration and phylogenetic uncertainties.     

Construction of hybrid gene libraries involving the circular permutation of DNA

The method of incremental truncation for the creation of hybrid enzymes (ITCHY) allows the creation of comprehensive fusion libraries between 5 and 3 fragments of two genes in a manner that is independent of DNA sequence homology. A methodology is presented for the creation of ITCHY libraries called circularly permuted ITCHY (CP-ITCHY) that allows the creation of ITCHY libraries in a manner that does not require extensive time point sampling. In addition, CP-ITCHY requires only a single vector and productively biases the library towards those fusions that are approximately the same size as the original genes. In the model system of creating fusions between fragments of the Escherichia coli and human glycinamide ribonucleotide transformylase genes, the CP-ITCHY libraries are shown to contain a diverse set of active fusions including those in regions of low-homology. In addition, a high percentage of active fusions were temperature-sensitive as they complemented an auxotrophic strain of Escherichia coli at 22 °C but not at 37 °C.     

Theoretical distribution of truncation lengths in incremental truncation libraries

Incremental truncation is a method for constructing libraries of every one base pair truncation of a segment of DNA. Incremental truncation libraries can be created using a time-dependent nuclease method or through the incorporation of alpha-phosphothioate dNTPs by PCR or by primer extension (THIO(pcr) truncation and THIO(extension) truncation, respectively). Libraries created by the fusion of two truncation libraries, known as ITCHY libraries, can be created using the above methods or by the incremental truncation-like method SHIPREC. Knowing and being able to tailor the distribution of truncations in incremental truncation, ITCHY and SHIPREC libraries would be beneficial for their use in protein engineering and other applications. However, the experimental determination of the distributions would require extensive, cost-prohibitive, DNA sequencing to obtain statistically relevant data. Instead, a theoretical prediction of the distributions was developed. Time-dependent incremental truncation libraries had the most uniform distribution of truncation lengths, but were biased against longer truncations. Essentially uniform distribution over the desired truncation range (from zero to N(max) base pairs) required that truncations be prepared up to at least 1.2-1.5 N(max). THIO(pcr) and THIO(extension) truncation libraries had a very nonuniform distribution of truncation lengths with a bias against longer truncations. Such nonuniformity could be significantly diminished by decreasing the incorporation rate of alphaS-dNTPs but at the expense of having a large fraction of the DNA truncated beyond the desired range or completely degraded. ITCHY libraries created using time-dependent truncation had the most uniform distribution of possible fusions and had the highest fraction of the library being parental-length fusions. However, the distribution of parental-length fusions was biased against fusions near the beginning/ends of genes unless the truncation libraries are prepared with a uniform distribution up to N(max). In contrast, SHIPREC libraries and THIO(pcr) ITCHY libraries, by the very nature of the nonuniform distributions of the truncated DNA, are ensured of having a uniform distribution of fusion points in parental-length fusions. This comes at the expense of having a smaller fraction of the library being parental-length fusions; however, this limitation can be overcome by performing size selection on the library.     

Multitasking with molecular dynamics Typhoon: quantifying nucleic acids and autoradiographs

With increased sensitivity and specificity, fluorescent assays are rapidly becoming the method of choice for nucleic acid quantification. The utility of the Typhoon scanner has now been extended to accurately measure low levels of DNA and RNA (5 ng ml^–1) with PicoGreen and RiboGreen dyes. In addition, with a few simple modifications, autoradiographic film images can be scanned and quantified with the Typhoon series of scanners.