Localization of interferon-induced lupus inclusions demonstrated by computer image reconstruction of monensin-treated Daudi cells.

A structural analysis of cells that contained the interferon-alpha-induced lupus inclusions (LI) was performed using a high-voltage electron microscope to determine the exact cellular location of LI and their association with normal cell organelles. LI were induced in the human B lymphoblastoid cell line, Daudi, by culturing with the pure recombinant human leukocyte interferon, IFLrA. Just prior to harvesting, a portion of the cells was treated with monensin to selectively swell the Golgi apparatus, and thereby simplify their identification using the electron microscope. Organellar associations between LI and the outer nuclear envelope and Golgi apparatus were identified in stereopairs of 1-micron sections prepared from both cells that were not treated with monensin and those that were treated with monensin. Serial 0.25-micron sections of the monensin-treated cells were prepared, and seven arbitrarily chosen cells were examined. Each of these cells contained a single LI, and it formed throughout an endoplasmic-reticulum region that made contact with both the outer nuclear envelope and the Golgi vesicles. Reconstruction of a cell by computer from the digitized negatives of serial sections clearly illustrated these relationships. This study reports the first determination of the association between LI and the Golgi apparatus. It also identifies the presence of only one LI in every cell, and the routine association of the LI with both the outer nuclear envelope and the Golgi apparatus. The unique cell location of LI formation suggests their functioning in membrane biogenesis, the trafficking of proteins to the plasma membrane or to cytoplasmic vesicles, or the processing of proteins for secretion.     

Denture repairs in different regions of Croatia in relation to prosthodontic teams

The purpose of this paper is to evaluate the incidence of denture repairs in different districts of Croatia through the year of 2002. and to analyse the percentage of different repairs (relinings, simple repairs up to 2 elements and complicated repairs-more than 2 elements) in relation to prosthodontic teams. Data on the number of dentures, and the number and types of denture repairs delivered in the Croatian regions of Zagreb, Rijeka, Split and Karlovac were obtained from the Croatian Institute for Health Insurance for the whole of the year 2002. Information of the number of prosthodontic teams operating in those regions was also obtained. Proportionally more denture repairs were carried out in Karlovac (18%) than Split (5%). The smallest percantage of dentures that required relining was registered in Split and the highest in Rijeka (chi2 = 36.7, p < 0.01). The smallest percentage of simple repairs was registered in Rijeka and the highest in Split (chi2 = 24.3, p < 0.01). The smallest percentage of complicated repairs was registered in Split and the highest in Karlovac. In each region the proportion of denture repairs and types of repairs were correlated with a number of prosthodontic teams in that region. Karlovac had the smallest percentage of specialistic prosthodontic teams and the highest rate of denture repairs.     

Dynamic imaging of protease activity with fluorescently quenched activity-based probes

Protease activity is tightly regulated in both normal and disease conditions. However, it is often difficult to monitor the dynamic nature of this regulation in the context of a live cell or whole organism. To address this limitation, we developed a series of quenched activity-based probes (qABPs) that become fluorescent upon activity-dependent covalent modification of a protease target. These reagents freely penetrate cells and allow direct imaging of protease activity in living cells. Targeted proteases are directly identified and monitored biochemically by virtue of the resulting covalent tag, thereby allowing unambiguous assignment of protease activities observed in imaging studies. We report here the design and synthesis of a selective, cell-permeable qABP for the study of papain-family cysteine proteases. This probe is used to monitor real-time protease activity in live human cells with fluorescence microscopy techniques as well as standard biochemical methods.     

Detection and quantitation of colored deposit-forming <Emphasis Type="Italic">Meiothermus</Emphasis> spp. in paper industry processes and end products

Colored biofilms cause problems in paper industry. In this work we used real-time PCR to detect and to quantitate members of the genus Meiothermus from the process samples and end products from 24 machines manufacturing pulp, paper and board in four countries. The results obtained from 200 samples showed the importance of members of the genus Meiothermus as ubiquitous biofoulers in paper machines. This genus was the dominant biofouler in some mills. From ≤10⁴ to 10¹¹ copies of Meiothermus 16S rRNA genes were found per gram of process deposit (wet weight). Meiothermus spp. were found in paper and board products with colored defects and connection between deposit-forming microbes and end-product spots was shown. 16S rRNA gene sequences of 29 biofilm producing bacterial isolates from different mills were determined. Based on sequence data, 25 of the isolates were assigned to the genus Meiothermus, with Meiothermus silvanus and M. ruber as the most frequent species.     

Evaluation of genotoxic effects of Apitol (cymiazole hydrochloride) in vitro by measurement of sister chromatid exchange

Apitol^®, with cymiazole hydrochloride as the active ingredient, is used in bee-keeping against the ectoparasitic mite Varroa destructor. The preparation was evaluated for genotoxicity in cultured human peripheral blood lymphocytes. Sister chromatid exchange, the mitotic index and the cell proliferation index were determined for three experimental concentrations of Apitol^® (0.001, 0.01 and 0.1 mg/ml). All concentrations significantly (p < 0.001) increased the mitotic index (MI = 7.35 ± 0.18%, 8.31 ± 0.20% and 12.33 ± 0.25%, respectively), the proliferative index (PI = 1.83 ± 0.01, 1.84 ± 0.01 and 1.88 ± 0.02, respectively) and the frequency of sister chromatid exchange (SCE = 8.19 ± 1.81, 8.78 ± 1.80 and 13.46 ± 1.88, respectively), suggesting that cymiazole hydrochloride has genotoxic potential.     

Autoradiographic study of protein and RNA formation during early development of Drosophila eggs

Permeabilized eggs of Drosophila melanogaster were incubated in tritiated uridine, valine, and phenylalanine. The uptake and incorporation into TCA-insoluble material were measured by scintillation counting. There was very little incorporation of uridine before the blastoderm stage. At the blastoderm stage, the egg took up 2.4 pmoles/hr of uridine and incorporated 0.13 pmoles into RNA (assuming no dilution of specific activity of the precursor). The uptake of amino acids varied with the age of the embryo; virgin eggs synthesized about as much protein as fertilized eggs. Autoradiography of eggs incubated in uridine showed a lack of RNA synthesis in nuclei until the start of the blastoderm formation. The small amount of uridine incorporation before this stage was due to mitochondria. Incorporation of amino acids was uniform in the cytoplasm until the blastoderm; there was no incorporation by yolk granules. Regional difference in labeling appeared during gastrulation. The pole cells did not form RNA during the blastoderm stage, formation started during gastrulation. Protein labeling of the pole cells, on the contrary, was very strong in the blastoderm and early gastrula. These results indicate that the expression of zygotic genome before the blastoderm stage is unlikely.     

Discovery of Triterpenoids as Reversible Inhibitors of α/β-hydrolase Domain Containing 12 (ABHD12)

Background

α/β-hydrolase domain containing (ABHD)12 is a recently discovered serine hydrolase that acts in vivo as a lysophospholipase for lysophosphatidylserine. Dysfunctional ABHD12 has been linked to the rare neurodegenerative disorder called PHARC (polyneuropathy, hearing loss, ataxia, retinosis pigmentosa, cataract). In vitro, ABHD12 has been implicated in the metabolism of the endocannabinoid 2-arachidonoylglycerol (2-AG). Further studies on ABHD12 function are hampered as no selective inhibitor have been identified to date. In contrast to the situation with the other endocannabinoid hydrolases, ABHD12 has remained a challenging target for inhibitor development as no crystal structures are available to facilitate drug design.

Methodology/Principal Findings

Here we report the unexpected discovery that certain triterpene-based structures inhibit human ABHD12 hydrolase activity in a reversible manner, the best compounds showing submicromolar potency. Based on structure activity relationship (SAR) data collected for 68 natural and synthetic triterpenoid structures, a pharmacophore model has been constructed. A pentacyclic triterpene backbone with carboxyl group at position 17, small hydrophobic substituent at the position 4, hydrogen bond donor or acceptor at position 3 accompanied with four axial methyl substituents was found crucial for ABHD12 inhibitor activity. Although the triterpenoids typically may have multiple protein targets, we witnessed unprecedented selectivity for ABHD12 among the metabolic serine hydrolases, as activity-based protein profiling of mouse brain membrane proteome indicated that the representative ABHD12 inhibitors did not inhibit other serine hydrolases, nor did they target cannabinoid receptors.

Conclusions/Significance

We have identified reversibly-acting triterpene-based inhibitors that show remarkable selectivity for ABHD12 over other metabolic serine hydrolases. Based on SAR data, we have constructed the first pharmacophore model of ABHD12 inhibitors. This model should pave the way for further discovery of novel lead structures for ABHD12 selective inhibitors.     

Continuous representations of time-series gene expression data.

We present algorithms for time-series gene expression analysis that permit the principled estimation of unobserved time points, clustering, and dataset alignment. Each expression profile is modeled as a cubic spline (piecewise polynomial) that is estimated from the observed data and every time point influences the overall smooth expression curve. We constrain the spline coefficients of genes in the same class to have similar expression patterns, while also allowing for gene specific parameters. We show that unobserved time points can be reconstructed using our method with 10-15% less error when compared to previous best methods. Our clustering algorithm operates directly on the continuous representations of gene expression profiles, and we demonstrate that this is particularly effective when applied to nonuniformly sampled data. Our continuous alignment algorithm also avoids difficulties encountered by discrete approaches. In particular, our method allows for control of the number of degrees of freedom of the warp through the specification of parameterized functions, which helps to avoid overfitting. We demonstrate that our algorithm produces stable low-error alignments on real expression data and further show a specific application to yeast knock-out data that produces biologically meaningful results.     

Risk factor analysis and diagnoses of coronary heart disease in patients with hypercholesterolemia from Croatian Zagorje County

Our aim is to determine if there exists a difference in risk factors and diagnosis between patients being treated on internal medicine ward for coronary heart disease who have higher levels of cholesterol in their blood and other patients, without proved higher levels of cholesterol, hospitalized for coronary heart disease. We followed patients hospitalized in General Hospital Zabok for coronary heart disease for the period between 2004-2006y. On admission patients were diagnosed with coronary heart disease based on laboratory markers specific for the disease (CK, troponin, LDH,CRP), ECG and history taking. We analyzed two groups of patients for diagnosis and risk factors on discharge from the hospital: one group with proven hypercholesterolemia, the other with coronary heart disease without hypercholesterolemia. For the duration of the study there were no significant alternations concerning risk factors for coronary heart disease, and hypertension was the most prevalent of these factors in both groups. Values of HDL, as an indirect indicator of coronary heart disease, were lower in both groups for the duration of the study. In group of patients with hypercholesterolemia myocardial infarction with a ST segment elevation, as a discharge diagnosis, was a more prevalent complication of the disease, while for the group of patients without hypercholesterolemia stable angina pectoris was more prevalent and this is explained as atheroma plaque stabilization when there are normal values of blood cholesterol.     

Insect pest management in the age of synthetic biology

Arthropod crop pests are responsible for 20% of global annual crop losses, a figure predicted to increase in a changing climate where the ranges of numerous species are projected to expand. At the same time, many insect species are beneficial, acting as pollinators and predators of pest species. For thousands of years, humans have used increasingly sophisticated chemical formulations to control insect pests but, as the scale of agriculture expanded to meet the needs of the global population, concerns about the negative impacts of agricultural practices on biodiversity have grown. While biological solutions, such as biological control agents and pheromones, have previously had relatively minor roles in pest management, biotechnology has opened the door to numerous new approaches for controlling insect pests. In this review, we look at how advances in synthetic biology and biotechnology are providing new options for pest control. We discuss emerging technologies for engineering resistant crops and insect populations and examine advances in biomanufacturing that are enabling the production of new products for pest control.