Dynamics of Human Replication Protein A Subunit Distribution and Partitioning in the Cell Cycle

Human replication protein A (RPA) is a three-subunit protein complex involved in DNA replication, repair, and recombination. To gain insight into the dynamics of subunit assembly, we examined the subcellular distribution of RPA subunits (p70, p34, and p11) during the cell cycle. All three subunits colocalized in G1 and S phases, showing a diffuse nuclear distribution in G1 but a dot-like nuclear pattern in S phase. During S phase, the subunits showed a pattern reminiscent of the replication granules/factories described by others as sites of replication machinery. In metaphase, p70 preferentially associated with the spindle poles, p34 was found on chromosomes, and p11 remained in the cytoplasm. In telophase, p70 and p34 appeared in the forming daughter nuclei; p11 remained in the cytoplasm until G1. Among the three subunits only p34 was associated with the nuclear matrix and this association persisted throughout the cell cycle. We conclude that (i) RPA complex assembly is differentially regulated, (ii) the replication machinery may be anchored to the nuclear matrix, and (iii) RPA subunits partition during mitosis and sort into daughter nuclei by different routes.     

Expression of functional TRPA1 receptor on human lung fibroblast and epithelial cells

The transient receptor potential subfamily A member 1 (TRPA1) is a non-selective cation channel implicated in the pathogenesis of several airway diseases like asthma and chronic obstructive pulmonary disease (COPD). Most of the research on TRPA1 focuses on its expression and function in neuronal context; studies investigating non-neuronal expression of TRPA1 are lacking. In the present study, we show functional expression of TRPA1 in human lung fibroblast cells (CCD19-Lu) and human pulmonary alveolar epithelial cell line (A549). We demonstrate TRPA1 expression at both mRNA and protein levels in these cell types. TRPA1 selective agonists like allyl isothiocyanate (AITC), 4-hydroxynonenal (4-HNE), crotonaldehyde and zinc, induced a concentration-dependent increase in Ca+2 influx in CCD19-Lu and A549 cells. AITC-induced Ca+2 influx was inhibited by Ruthenium red (RR), a TRP channel pore blocker, and by GRC 17536, a TRPA1 specific antagonist. Furthermore, we also provide evidence that activation of the TRPA1 receptor by TRPA1 selective agonists promotes release of the chemokine IL-8 in CCD19-Lu and A549 cells. The IL-8 release in response to TRPA1 agonists was attenuated by TRPA1 selective antagonists. In conclusion, we demonstrate here for the first time that TRPA1 is functionally expressed in cultured human lung fibroblast cells (CCD19-Lu) and human alveolar epithelial cell line (A549) and may have a potential role in modulating release of this important chemokine in inflamed airways.     

High expression of Cyp6g1, a cytochrome P450 gene, does not necessarily confer DDT resistance in Drosophila melanogaster

Cytochrome P450 monooxygenases, a family of detoxifying enzymes, are thought to confer resistance to various insecticides including DDT. Daborn et al. [Daborn, P., Yen, J.L., Bogwitz, M., Le Goff, G., Feil, et al. 2002. A single p450 allele associated with insecticide resistance in Drosophila. Science 297, 2253-2256.] suggested that the Accord transposable element causes overexpression of a Cyp6g1 allele, which has spread globally and is the basis of DDT resistance in Drosophila melanogaster populations. To determine whether the same phenomenon also operates in other Drosophila strains, we investigated 91-R, 91-C, ry(506), Wisconsin, Canton-SH and Hikone-RH strains. While the LC(50) values for the 91-R and Wisconsin strains are 8348 microg and 447 microg of DDT, respectively, values for the other four strains range between 0.74 to 20.9 microg. As expected, the susceptible ry(506) and 91-C strains have about 16-33-fold lower levels of CYP6G1 mRNA than the resistant 91-R and Wisconsin strains. Surprisingly, CYP6G1 mRNA and protein levels in the Canton-SH and Hikone-RH strains are as high as in the two resistant strains, yet they are as susceptible as the 91-C strain. The susceptible phenotype of the Canton-SH and Hikone-RH strains is not due to mutation in the Cyp6g1 gene; sequence analysis showed that Cyp6g1 alleles of resistant and susceptible strains are very similar and cannot be classified into resistant and susceptible alleles. As observed by others, we also found that only the 5'-upstream DNA of overexpressing alleles of Cyp6g1 has an insertional DNA, which is similar to Accord and Ninja elements. To examine the role of Cyp6g1 in DDT resistance, we substituted the Cyp6g1 allele of the 91-R strain with the allele from the susceptible 91-C strain via recombination and synthesized three recombinant lines. All three lines lacked Accord insertion and showed low expression of Cyp6g1 like the 91-C strain, yet they were as highly resistant as the 91-R strain. We conclude a strain may not have to have Accord insertion in the Cyp6g1 gene and the Cyp6g1 itself may not have to be overexpressed for DDT resistance to occur.     

High density electromyography data of normally limbed and transradial amputee subjects for multifunction prosthetic control

Pattern recognition based control of powered upper limb myoelectric prostheses offers a means of extracting more information from the available muscles than conventional methods. By identifying repeatable patterns of muscle activity across multiple muscle sites rather than relying on independent EMG signals it is possible to provide more natural, reliable control of myoelectric prostheses. The purposes of this study were to (1) determine if participants can perform distinctive muscle activation patterns associated with multiple wrist and hand movements reliably and (2) to show that high density EMG can be applied individually to determine the electrode location of a clinically acceptable number of electrodes (maximally eight) to classify multiple wrist and hand movements reliably in transradial amputees. Eight normally limbed subjects (five female, three male) and four transradial amputee subjects (two traumatic and congenital) subjects participated in this study, which examined the classification accuracies of a pattern recognition control system. It was found that tasks could be classified with high accuracy (85-98%) with normally limbed subjects (10-13 tasks) and with amputees (4-6) tasks. In healthy subjects, reducing the number of electrodes to eight did not affect accuracy significantly when those electrodes were optimally placed, but did reduce accuracy significantly when those electrodes were distributed evenly. In the amputee subjects, reducing the number of electrodes up to 4 did not affect classification accuracy or the number of tasks with high accuracy, independent of whether those remaining electrodes were evenly distributed or optimally placed. The findings in healthy subjects suggest that high density EMG testing is a useful tool to identify optimal electrode sites for pattern recognition control, but its use in amputees still has to be proven. Instead of just identifying the electrode sites where EMG activity is strong, clinicians will be able to choose the electrode sites that provide the most important information for classification.     

The walnut (Juglans regia) genome sequence reveals diversity in genes coding for the biosynthesis of non‐structural polyphenols

The Persian walnut (Juglans regia L.), a diploid species native to the mountainous regions of Central Asia, is the major walnut species cultivated for nut production and is one of the most widespread tree nut species in the world. The high nutritional value of J. regia nuts is associated with a rich array of polyphenolic compounds, whose complete biosynthetic pathways are still unknown. A J. regia genome sequence was obtained from the cultivar ‘Chandler’ to discover target genes and additional unknown genes. The 667‐Mbp genome was assembled using two different methods (SOAPdenovo2 and MaSuRCA), with an N50 scaffold size of 464 955 bp (based on a genome size of 606 Mbp), 221 640 contigs and a GC content of 37%. Annotation with MAKER‐P and other genomic resources yielded 32 498 gene models. Previous studies in walnut relying on tissue‐specific methods have only identified a single polyphenol oxidase (PPO) gene (JrPPO1). Enabled by the J. regia genome sequence, a second homolog of PPO (JrPPO2) was discovered. In addition, about 130 genes in the large gallate 1‐β‐glucosyltransferase (GGT) superfamily were detected. Specifically, two genes, JrGGT1 and JrGGT2, were significantly homologous to the GGT from Quercus robur (QrGGT), which is involved in the synthesis of 1‐O‐galloyl‐β‐d ‐glucose, a precursor for the synthesis of hydrolysable tannins. The reference genome for J. regia provides meaningful insight into the complex pathways required for the synthesis of polyphenols. The walnut genome sequence provides important tools and methods to accelerate breeding and to facilitate the genetic dissection of complex traits.     

MICRONUCLEAR RIBONUCLEIC ACID IN TETRAHYMENA PYRIFORMIS

The presence of RNA in the micronucleus of Tetrahymena pyriformis was detected by electron microscope radioautography after incubation with tritiated precursors. The specificity of RNA labeling was shown by ribonuclease digestion. The period of appearance of labeled RNA in the micronucleus is approximately coincident with the DNA synthesis period for the micronucleus. Pulse-chase experiments showed that the micronuclear RNA disappears during the interphase period. The experiments do not distinguish whether the micronuclear RNA is synthesized in situ or acquired by migration from the macronucleus. In either case it is notable that the appearance of labeled RNA is detected in the micronucleus only during the micronuclear S phase.     

Impact of depth filtration on disulfide bond reduction during downstream processing of monoclonal antibodies from CHO cell cultures

Monoclonal antibody interchain disulfide bond reduction was observed in a Chinese Hamster Ovary manufacturing process that used single-use technologies. A similar reduction has been reported for processes that involved high mechanical shear recovery unit operations, such as continuous flow centrifugation and when the clarified harvest was stored under low dissolved oxygen (DO) conditions (Trexler-Schmidt et al., 2010. Biotechnology and Bioengineering, 106(3), 452–461). The work described here identifies disposable depth filtration used during cell culture harvest operations as a shear-inducing unit operation causing cell lysis. As a result, reduction of antibody interchain disulfide bonds was observed through the same mechanisms described for continuous flow centrifugation. Small-scale depth-filtration models were developed, and the differential pressure (Δ P) of the primary depth filter was identified as the key factor contributing to cell lysis. Strong correlations of Δ P and cell lysis were generated by measuring the levels of lactate dehydrogenase and thiol in the filtered harvest material. A simple risk mitigation strategy was implemented during manufacturing by providing an air overlay to the headspace of a single-use storage bag to maintain sufficient DO in the clarified harvest. In addition, enzymatic characterization studies determined that thioredoxin reductase and glucose-6-phosphate dehydrogenase are critical enzymes involved in antibody reduction in a nicotinamide adenine dinucleotide phosphate (NADP ⁺)/NADPH-dependent manner.     

The interaction of mycobacterial protein Rv2966c with host chromatin is mediated through non-CpG methylation and histone H3/H4 binding

To effectively modulate the gene expression within an infected mammalian cell, the pathogen Mycobacterium tuberculosis would need to bring about epigenetic modifications at appropriate genomic loci. Working on this hypothesis, we show in this study that the mycobacterial protein Rv2966c is a 5-methylcytosine-specific DNA methyltransferase that is secreted out from the mycobacterium and gets localized to the nucleus in addition to the cytoplasm inside the host cell. Importantly, Rv2966c binds to specific DNA sequences, methylates cytosines predominantly in a non-CpG context and its methylation activity is positively influenced by phosphorylation. Interestingly, like the mammalian DNA methyltransferase, DNMT3L, Rv2966c can also interact with histone proteins. Ours is the first study that identifies a protein from a pathogenic bacteria with potential to influence host DNA methylation in a non-canonical manner providing the pathogen with a novel mechanism to alter the host epigenetic machinery. This contention is supported by repression of host genes upon M. tuberculosis infection correlated with Rv2966c binding and non-CpG methylation.