In vitro binding of [14C]2,5-hexanedione to rat neuronal cytoskeletal proteins

2,5-Hexanedione (2,5-HD) induces central-peripheral axonpathy characterized by the accumulation of 10-nm neurofilaments proximal to the nodes of Ranvier and a Wallerian-type degeneration. It has been postulated that neurofilament crosslinking may be involved in the production of this axonopathy. A potential initiating event in this neurotoxic process may be the direct binding of 2,5-HD to neurofilament and microtubule proteins. In this study, the in vitro binding of [¹⁴C]2,5-HD to neurofilament and microtubule proteins was examined. Neurofilament proteins isolated from rat spinal cord or microtubule proteins isolated from rat brain were incubated in the presence of 2,5-HD at concentrations ranging 25 to 500 mM. Quantitative analysis of sodium dodecyl sulfate (SDS) polyacrylamide gels revealed a dose- and time-dependent binding of 2,5-HD to both neurofilament proteins and microtubule proteins. Expressed as pmol 2,5-HD bound per g protein, the observed relative binding was MAP2>NF160>NF200>NF68>tubulin. These data demonstrate the direct binding of 2,5-HD to cytoskeletal proteins including both neurofilaments and microtubules.     

The influence of scald water pH on the death rates of Salmonella typhimurium and other bacteria attached to chicken skin

Factory trials where scald tank water was maintained at pH 9.0 +/- 0.2 showed that compared with the usual system of scalding when the water is at pH 6.0 for much of the working day the bacterial counts on carcases post scalding and plucking were significantly lower. In laboratory experiments, attached Salmonella typhimurium and the naturally occurring skin flora were found to be killed significantly more quickly in water at pH 9.0 +/- 0.2.     

Application of an optimized marker amplification protocol in immunohistochemistry — a valuable tool for visualizing antigenic sites of low signal strength or sparse distribution

Amplification of immunohistochemical markers received considerable attention during the 1980s and 1990s. The amplification approach was largely abandoned following the development of antigen retrieval and reporter amplification techniques, because the latter were incorporated more easily into high throughput automated procedures in industrial and diagnostic laboratories. There remain, however, a number of instances where marker amplification still has much to offer. Consequently, we examined experimentally the utility of an optimized marker amplification technique in diagnostically relevant tissue where either the original signal strength was low or positive sites were visible, but sparsely distributed. Marker amplification in the former case not only improved the visibility of existing positive sites, but also revealed additional sites that previously were undetectable. In the latter case, positive sites were rendered more intense and therefore more easily seen during low magnification examination of large areas of tissue.     

EFFECT OF VITAMIN E SUPPLEMENTATION AND PARTIAL SUBSTITUTION OF POLY- WITH MONO-UNSATURATED FATTY ACIDS IN PIG DIETS ON MUSCLE,AND MICROSOME EXTRACT a-TOCOPHEROL CONCENTRATION AND LIPID OXIDATION

The experiment was organized in a 3×2 factorial arrangement with three dietary fat blends and a basal (20 mg kg^?1 diet) or supplemented (220 mg kg^?1) level of α-tocopheryl acetate. Dietary vitamin E and monounsaturated to polyunsaturated fatty acid ratio (dietary MUFA/PUFA) affected muscle α-tocopherol concentration (α-tocopherol [log μg g^?1]=0.18 (±0.105)+0.0034 (±0.0003)·dietary α-tocopherol [mg kg^?1 diet] (P<0.0001)+0.39 (±0.122)·dietary MUFA/PUFA (P<0.0036)). An interaction between dietary α-tocopherol and dietary MUFA/PUFA exists for microsome α-tocopherol concentration (α-tocopherol [log μg g^?1]=1.14 (±0.169) (P<0.0001)+0.0056 (±0.00099)·dietary α-tocopherol [mg kg^?1 diet] (P<0.0001)+0.54 (±0.206)·dietary MUFA/PUFA (P<0.0131)?0.0033 (±0.0011)·dietary α-tocopherol [mg kg^?1)]×dietary MUFA/PUFA (P<0.0067)), and hexanal concentration in meat (hexanal [ng·g^?1]=14807.9 (±1489.8)?28.8 (±10.6) dietary α-tocopherol [mg·kg^?1] (P<0.01)?8436.6 (±1701.6)·dietary MUFA/PUFA (P<0.001)+24.0 (±11.22)·dietary α-tocopherol·dietary MUFA/PUFA (P<0.0416)). It is concluded that partial substitution of dietary PUFA with MUFA lead to an increase in the concentration of α-tocopherol in muscle and microsome extracts. An interaction between dietary α-tocopherol and fatty acids exists, in which at low level of dietary vitamin E inclusion, a low MUFA/PUFA ratio leads to a reduction in the concentration of α-tocopherol in microsome extracts and a concentration of hexanal in meat above the expected values.     

Quirks of dye nomenclature. 5. Rhodamines

Rhodamines were first produced in the late 19^th century, when they constituted a new class of synthetic dyes. These compounds since have been used to color many things including cosmetics, inks, textiles, and in some countries, food products. Certain rhodamine dyes also have been used to stain biological specimens and currently are widely used as fluorescent probes for mitochondria in living cells. The early history and current biological applications are sketched briefly and an account of the ambiguities, complications and confusions concerning dye identification and nomenclature are discussed.     

Quirks of dye nomenclature. 6. Malachite green

Malachite green was discovered independently by two researchers in Germany in the 19^th century and found immediate employment as a dye and a pigment. Subsequently, other uses, such as staining biological specimens, emerged. A much later application was the control of fungal and protozoan infections in fish, for which the dye remains popular, although illegal in many countries owing to a variety of toxicity problems. In solution, malachite green can exist as five different species depending on the pH. The location of the positive charge of the colored cation on a carbon atom or a nitrogen atom is still debated. The original names of this dye, and their origins, are briefly surveyed.     

Quantitative variability of 342 plasma proteins in a human twin population

The degree and the origins of quantitative variability of most human plasma proteins are largely unknown. Because the twin study design provides a natural opportunity to estimate the relative contribution of heritability and environment to different traits in human population, we applied here the highly accurate and reproducible SWATH mass spectrometry technique to quantify 1,904 peptides defining 342 unique plasma proteins in 232 plasma samples collected longitudinally from pairs of monozygotic and dizygotic twins at intervals of 2–7 years, and proportioned the observed total quantitative variability to its root causes, genes, and environmental and longitudinal factors. The data indicate that different proteins show vastly different patterns of abundance variability among humans and that genetic control and longitudinal variation affect protein levels and biological processes to different degrees. The data further strongly suggest that the plasma concentrations of clinical biomarkers need to be calibrated against genetic and temporal factors. Moreover, we identified 13 cis‐SNPs significantly influencing the level of specific plasma proteins. These results therefore have immediate implications for the effective design of blood‐based biomarker studies.     

Impact of QTL properties on the accuracy of multi-breed genomic prediction

Background

Although simulation studies show that combining multiple breeds in one reference population increases accuracy of genomic prediction, this is not always confirmed in empirical studies. This discrepancy might be due to the assumptions on quantitative trait loci (QTL) properties applied in simulation studies, including number of QTL, spectrum of QTL allele frequencies across breeds, and distribution of allele substitution effects. We investigated the effects of QTL properties and of including a random across- and within-breed animal effect in a genomic best linear unbiased prediction (GBLUP) model on accuracy of multi-breed genomic prediction using genotypes of Holstein-Friesian and Jersey cows.

Methods

Genotypes of three classes of variants obtained from whole-genome sequence data, with moderately low, very low or extremely low average minor allele frequencies (MAF), were imputed in 3000 Holstein-Friesian and 3000 Jersey cows that had real high-density genotypes. Phenotypes of traits controlled by QTL with different properties were simulated by sampling 100 or 1000 QTL from one class of variants and their allele substitution effects either randomly from a gamma distribution, or computed such that each QTL explained the same variance, i.e. rare alleles had a large effect. Genomic breeding values for 1000 selection candidates per breed were estimated using GBLUP modelsincluding a random across- and a within-breed animal effect.

Results

For all three classes of QTL allele frequency spectra, accuracies of genomic prediction were not affected by the addition of 2000 individuals of the other breed to a reference population of the same breed as the selection candidates. Accuracies of both single- and multi-breed genomic prediction decreased as MAF of QTL decreased, especially when rare alleles had a large effect. Accuracies of genomic prediction were similar for the models with and without a random within-breed animal effect, probably because of insufficient power to separate across- and within-breed animal effects.

Conclusions

Accuracy of both single- and multi-breed genomic prediction depends on the properties of the QTL that underlie the trait. As QTL MAF decreased, accuracy decreased, especially when rare alleles had a large effect. This demonstrates that QTL properties are key parameters that determine the accuracy of genomic prediction.

Electronic supplementary material

The online version of this article (doi:10.1186/s12711-015-0124-6) contains supplementary material, which is available to authorized users.     

The Rac1 C-terminal polybasic region regulates the nuclear localization and protein degradation of Rac1

We observed evolutionary conservation of canonical nuclear localization signal sequences (K(K/R)X(K/R)) in the C-terminal polybasic regions (PBRs) of some Rac and Rho isoforms. Canonical D-box sequences (RXXL), which target proteins for proteasome-mediated degradation, are also evolutionarily conserved near the PBRs of these small GTPases. We show that the Rac1 PBR (PVKKRKRK) promotes Rac1 nuclear accumulation, whereas the RhoA PBR (RRGKKKSG) keeps RhoA in the cytoplasm. A mutant Rac1 protein named Rac1 (pbrRhoA), in which the RhoA PBR replaces the Rac1 PBR, has greater cytoplasmic localization, enhanced resistance to proteasome-mediated degradation, and higher protein levels than Rac1. Mutating the D-box by substituting alanines at amino acids 174 and 177 significantly increases the protein levels of Rac1 but not Rac1(pbrRhoA). These results suggest that Rac1 (pbrRhoA) is more resistant than Rac1 to proteasome-mediated degradative pathways involving the D-box. The cytoplasmic localization of Rac1(pbrRhoA) provides the most obvious reason for its resistance to proteasome-mediated degradation, because we show that Rac1(pbrRhoA) does not greatly differ from Rac1 in its ability to stimulate membrane ruffling or to interact with SmgGDS and IQGAP1-calmodulin complexes. These findings support the model that nuclear localization signal sequences in the PBR direct Rac1 to the nucleus, where Rac1 participates in signaling pathways that ultimately target it for degradation.