Beta B1 crystallin is an amine-donor substrate for tissue transglutaminase

The effects of tissue transglutaminase on the water-soluble proteins in bovine lens homogenates are described. Addition of liver transglutaminase and Ca2+ to calf lens homogenates resulted not only in the appearance of 50- and 57-kDa dimers, but also in a decrease in the amount of beta B1 crystallin and the almost complete disappearance of beta B3 and beta A3. This is not the result of Ca2+-induced proteolysis, since histamine completely inhibits this phenomenon. It may be concluded that these polypeptides are involved in beta-crystallin crosslinking by transglutaminase. This notion was confirmed by using beta B1- and beta Bp-specific antisera. Both sera reacted with the 57-kDa dimer; the beta Bp-specific antiserum also reacted with the 50-kDa dimer. No reaction in the region 50-57 kDa was detectable when EDTA was used instead of Ca2+. Using reconstituted mixtures of beta B1- and beta Bp-crystallin chains, and N-terminally truncated derivatives thereof, it was shown that in the beta B1/beta Bp dimer, glutamine residue -9 of beta Bp crosslinks to one of the lysine residues in the N-terminal extension of beta B1.     

Development of cell surface saccharides on embryonic pancreatic cells

Using a battery of seven lectin-ferritin conjugates as probes for cell surface glycoconjugates, we have studied the pattern of plasmalemmal differentiation of cells in the embryonic rat pancreas from day 15 in utero to the early postpartum stage. Our results indicate that differentiation of plasmalemmal glycoconjugates on acinar, endocrine, and centroacinar cells is temporally correlated with development and is unique for each cell type, as indicated by lectin-ferritin binding. Specifically, (a) expression of adult cell surface saccharide phenotype can be detected on presumptive acinar cells as early as 15 d in utero, as indicated by soybean agglutinin binding, and precedes development of intracellular organelles characteristic of mature acinar cells; (b) maturation of the plasmalemma of acinar cells is reached after intracellular cytodifferentiation is completed, as indicated by appearance of Con A and fucoselectin binding sites only at day 19 of development; conversely, maturation of the endocrine cell plasmalemma is accompanied by "loss" (masking) of ricinus communis II agglutinin receptors; and (c) binding sites for fucose lectins and for soybean agglutinin are absent on endocrine and centroacinar cells at all stages examined. We conclude that acinar, centroacinar, and endocrine cells develop from a common progenitor cell(s) whose plasmalemmal carbohydrate composition resembles most closely that of the adult centroacinar cell. Finally, appearance of acinar lumina beginning at approximately 17 d in utero is accompanied by differenetiation of apical and basolateral plasmalemmal domains of epithelial cells, as indicated by enhanced binding of several lectin-ferritin conjugates to the apical plasmalemmal, a pattern that persists from this stage through adult life.     

Age-Specific Mortality During the 1918 Influenza Pandemic: Unravelling the Mystery of High Young Adult Mortality

The worldwide spread of a novel influenza A (H1N1) virus in 2009 showed that influenza remains a significant health threat, even for individuals in the prime of life. This paper focuses on the unusually high young adult mortality observed during the Spanish flu pandemic of 1918. Using historical records from Canada and the U.S., we report a peak of mortality at the exact age of 28 during the pandemic and argue that this increased mortality resulted from an early life exposure to influenza during the previous Russian flu pandemic of 1889–90. We posit that in specific instances, development of immunological memory to an influenza virus strain in early life may lead to a dysregulated immune response to antigenically novel strains encountered in later life, thereby increasing the risk of death. Exposure during critical periods of development could also create holes in the T cell repertoire and impair fetal maturation in general, thereby increasing mortality from infectious diseases later in life. Knowledge of the age-pattern of susceptibility to mortality from influenza could improve crisis management during future influenza pandemics.

“The war is over – and I must go” Egon Schiele, 1890–1918.

     

Interactive Visualization and Industrial Ecology: Applications,Challenges, and Opportunities

The emergence of increasingly complex data in industrial ecology (IE) has caused scholarly interest in interactive visualization (IV). IV allows users to interact with data, aiding in processing and interpreting complex datasets, processes, and simulations. Consequently, IV can help IE practitioners communicate the complexities of their methods and results, shed light on the underlying research assumptions, and enable more transparent monitoring of data quality and error. This can significantly increase the reach and impact of research, promote transparency, reproducibility, and open science, as well as improve the clarity and presentation of IE research. A review of current IV applications reveals that, while data exploration has received some attention among IE practitioners, IV applications in scientific communication are clearly lacking. With the help of a working example, we explore the value of IV, discuss its operationalization, and highlight challenges that the IE community must face during IV uptake. Such challenges include technical and knowledge limitations, limits on user interaction, and implementation strategies. With these challenges in mind, we outline key aspects needed to lift the IE field to the forefront of scientific communication in the coming years. Among these, we draft the basic principles of a “Hub for Interactive Visualization in Industrial Ecology” (HIVE), a point of encounter where IE practitioners could find an array of data visualization tools that are geared toward IE datasets. IV is here to stay, and its inceptive stage presents many opportunities to IE practitioners to shape its operationalization and benefit from early adoption.     

The cover of living and dead corals from airborne remote sensing

Trends in coral cover are widely used to indicate the health of coral reefs but are costly to obtain from field survey over large areas. In situ studies of reflected spectra at the coral surface show that living and recently dead colonies can be distinguished. Here, we investigate whether such spectral differences can be detected using an airborne remote sensing instrument. The Compact Airborne Spectrographic Imager (Itres Research Ltd, Canada) was flown in two configurations: 10 spectral bands with 1-m² pixels and 6 spectral bands with 0.25-m² pixels. First, we show that an instrument with 10 spectral bands possesses adequate spectral resolution to distinguish living Porites, living Pocillopora spp., partially dead Porites, recently dead Porites (total colony mortality within 6 months), old dead (>6 months) Porites, Halimeda spp., and coralline red algae when there is no water column to confuse spectra. All substrata were distinguished using fourth-order spectral derivatives around 538 nm and 562 nm. Then, at a shallow site (Tivaru) at Rangiroa Atoll, Tuamotu Archipelago (French Polynesia), we show that live and dead coral can be distinguished from the air to a depth of at least 4 m using first- and fourth-order spectral derivatives between 562–580 nm. However, partially dead and recently dead Porites colonies could not be distinguished from an airborne platform. Spectral differences among substrata are then exploited to predict the cover of reef substrata in ten 25-m² plots at nearby Motu Nuhi (max depth 8 m). The actual cover in these plots was determined in situ using quadrats with a 0.01-m² grid. Considerable disparity occurred between field and image-based measures of substrate cover within individual 25-m² quadrats. At this small scale, disparity, measured as the absolute difference in cover between field and remote-sensing methods, reached 25% in some substrata but was always less than 10% for living coral (99% of which consisted of Porites spp.). At the scale of the reef (all ten 25-m² quadrats), however, disparities in percent cover between imagery and field data were less than 10% for all substrata and extremely low for some classes (e.g. <3% for living Porites, recently dead Porites and Halimeda). The least accurately estimated substrata were sand and coralline red algae, which were overestimated by absolute values 7.9% and 6.6%, respectively. The precision of sampling was similar for field and remote-sensing methods: field methods required 19 plots to detect a 10% difference in coral cover among three reefs with a statistical power of 95%. Remote-sensing methods required 21 plots. However, it took 1 h to acquire imagery over 92,500 m² of reef, which represents 3,700 plots of 25 m² each, compared with 3 days to survey 10 such plots underwater. There were no significant differences in accuracy between 1-m² and 0.25-m² image resolutions, suggesting that the advantage of using smaller pixels is offset by reduced spectral information and an increase in noise (noise was observed to be 1.6–1.8 times greater in 0.25-m² pixels). We show that airborne remote sensing can be used to monitor coral and algal cover over large areas, providing that water is shallow and clear, and that brown fleshy macroalgae are scarce, that depth is known independently (e.g. from sonar survey).     

Role of platelet derived endothelial cell growth factor/thymidine phosphorylase in fluoropyrimidine sensitivity and potential role of deoxyribose-1-phosphate

Thymidine phosphorylase (TP) catalyzes the phosphorolytic cleavage of thymidine (TdR) to thymine and deoxyribose-1-phosphate (dR-1-P). TP, which is overexpressed in a wide variety of solid tumors, is involved in the activation and inactivation of fluoropyrimidines. We investigated the role of TP in 5'-deoxy-5-fluorouridine (5'DFUR), 5-fluorouracil (5FU) and trifluorothymidine (TFT) sensitivity. TP had no effect on TFT while it activated 5'DFUR and to a lesser extent 5FU. In order to provide an explanation for this difference in activation of 5'DFUR and 5FU, we studied the role of the 5FU co-substrate, dR-1-P, needed for its activation.     

Insulin inhibits extracellular regulated kinase 1/2 phosphorylation in a phosphatidylinositol 3-kinase (PI3) kinase-dependent manner in Neuro2a cells

Insulin signalling is well studied in peripheral tissue, but not in neuronal tissue. To gain more insight into neuronal insulin signalling we examined protein kinase B (PKB) and extracellular regulated kinase 1 and 2 (ERK1/2) regulation in serum-deprived Neuro2a cells. Insulin phosphorylated PKB in a dose-dependent manner but reduced phosphorylation of ERK1/2. Both processes were phosphatidylinositol 3-kinase (PI3K) dependent. Interestingly, blockade of PI3K in combination with insulin induced phosphorylation of ERK1/2. The phosphorylation of ERK1/2 could be blocked with a specific inhibitor of mitogen-activated protein/ERK kinase (MEK), suggesting that it was mediated through the highly conserved Ras-Raf-MEK-ERK1/2 pathway. Prolonged exposure to high concentrations of insulin resulted in a desensitized PI3K-PKB route. The insulin-induced inhibition of ERK1/2 phosphorylation was also diminished when the PI3K-PKB route was desensitized. Blockade of PI3K in combination with insulin, however, still resulted in an unaltered MEK-dependent phosphorylation of ERK1/2. We conclude that PI3K is an important integrator of insulin signalling in Neuro2a cells as it regulates activation of PKB and inhibition of ERK1/2, and is sensitive to the duration of the insulin stimulus.     

Inactivation of the RNase Activity of Glycoprotein Erns of Classical Swine Fever Virus Results in a Cytopathogenic Virus

Envelope glycoprotein E^rns of classical swine fever virus (CSFV) has been shown to contain RNase activity and is involved in virus infection. Two short regions of amino acids in the sequence of E^rns are responsible for RNase activity. In both regions, histidine residues appear to be essential for catalysis. They were replaced by lysine residues to inactivate the RNase activity. The mutated sequence of E^rns was inserted into the p10 locus of a baculovirus vector and expressed in insect cells. Compared to intact E^rns, the mutated proteins had lost their RNase activity. The mutated proteins reacted with E^rns-specific neutralizing monoclonal and polyclonal antibodies and were still able to inhibit infection of swine kidney cells (SK6) with CSFV, but at a concentration higher than that measured for intact E^rns. This result indicated that the conformation of the mutated proteins was not severely affected by the inactivation. To study the effect of these mutations on virus infection and replication, a CSFV mutant with an inactivated E^rns (FLc13) was generated with an infectious DNA copy of CSFV strain C. The mutant virus showed the same growth kinetics as the parent virus in cell culture. However, in contrast to the parent virus, the RNase-negative virus induced a cytopathic effect in swine kidney cells. This effect could be neutralized by rescue of the inactivated E^rns gene and by neutralizing polyclonal antibodies directed against E^rns, indicating that this effect was an inherent property of the RNase-negative virus. Analyses of cellular DNA of swine kidney cells showed that the RNase-negative CSFV induced apoptosis. We conclude that the RNase activity of envelope protein E^rns plays an important role in the replication of pestiviruses and speculate that this RNase activity might be responsible for the persistence of these viruses in their natural host.Classical swine fever virus (CSFV), bovine viral diarrhea virus (BVDV), and border disease virus belong to the genus Pestivirus within the family Flaviviridae (10). The viruses are structurally, antigenically, and genetically closely related. BVDV and border disease virus can infect ruminants and pigs. CSFV infections are restricted to pigs (6). Pestiviruses are small, enveloped, positive-stranded RNA viruses (23). The genome of pestiviruses varies in length from 12.5 to 16.5 kb (1, 2, 7, 17, 19, 25, 26, 28, 32) and contains a single large open reading frame (ORF) (1, 7, 8, 17, 26). The ORF is translated into a polyprotein which is processed into mature proteins by viral and host cell proteases (30). The envelope of the pestivirus virion contains three glycoproteins, E^rns, E1, and E2 (35). Animals infected with pestiviruses raise antibodies against at least two viral glycoproteins, namely, E^rns and E2 (16, 34, 42). Inhibition studies with E2 and E^rns produced in insect cells showed that both envelope proteins are indispensable for viral attachment and entry of pestiviruses into susceptible cells (13). In the virion, E^rns is present as a homodimer with a molecular mass of about 100 kDa (35). E^rns lacks a membrane anchor, and association with the envelope is accomplished by an as-yet-unknown mechanism. Significant amounts of E^rns are secreted from infected cells (30). A unique feature is that E^rns, besides being an envelope protein, possesses RNase activity (12, 31). E^rns belongs to the family of extracellular RNases consisting of several fungal (e.g., RNase T₂ and Rh) and plant (e.g., S glycoproteins of Nicotiana alata) RNases (12, 31). These RNases contain two homologous regions of 8 amino acids each which are spaced by 38 (E^rns) nonhomologous amino acids and which form the RNase active site. Histidine residues in both regions appear to be essential for RNase catalysis (15).The role of this RNase activity in the replication of pestiviruses or in the pathogenesis of a pestivirus infection is an interesting issue that, as yet, has not been studied. The availability of a recently generated infectious DNA copy of CSFV strain C (24) has given us the opportunity to study the effect of defined mutations in a pestivirus genome. In this paper, we report the inactivation of the RNase activity of E^rns by mutagenesis. To characterize the mutated proteins, we produced large amounts of them in insect cells (12). By reverse genetics, we generated an RNase-negative CSFV recombinant. The effect of the inactivation of the RNase activity of E^rns on the replication of CSFV in vitro was studied.