Resource partitioning of the host fungus Coriolus versicolor by two ciid beetles: the role of odour compounds and host ageing

The ciid beetles Octotemnus glabriculus and Cis boleti exploit different developmental stages of fruit bodies of their preferred host fungus Coriolus versicolor . Larvae of the smaller beetle, O. glabriculus , mainly use young, expanding, fruit bodies; adults of O. glabriculus are predominantly found in young fruit bodies. By contrast, adults and larvae of the larger beetle, C. boleti , are prevalent in fully developed fruit bodies of C. versicolor . Because fruit bodies of most genets emerge during spring and early summer and mature by autumn, O. glabriculus and C. boleti breed in separated seasons. Adults and larvae of O. glabriculus are abundant in spring and early summer. By contrast, the number of adults and larvae of C. boleti increases gradually from late spring to summer and peaks in autumn. We conducted a field experiment that suggests that the phenological dynamics of C. versicolor fruit bodies drive the separation of breeding seasons between O. glabriculus and C. boleti . Additionally, laboratory experiments revealed that O. glabriculus and C. boleti have differential behavioural responses to odour compounds from young and mature fruit bodies of C. versicolor . We conclude that age-related changes in the chemical composition of fruit bodies may allow O. glabriculus and C. boleti to discriminate among C. versicolor , thus providing a mechanism for the partitioning of the resource.     

The signal peptide peptidase SppA is involved in sterol regulatory element‐binding protein cleavage and hypoxia adaptation in Aspergillus nidulans

Using forward genetics, we revealed that the signal peptide peptidase (SPP) SppA, an aspartyl protease involved in regulated intramembrane proteolysis (RIP), is essential for hypoxia adaptation in Aspergillus nidulans, as well as hypoxia‐sensitive mutant alleles of a sterol regulatory element‐binding protein (SREBP) srbA and the Dsc ubiquitin E3 ligase complex dscA‐E. Both null and dead activity [D337A] mutants of sppA failed to grow in hypoxia, and the growth defect of ΔsppA was complemented by nuclear SrbA‐N381 expression. Additionally, SppA interacted with SrbA in the endoplasmic reticulum, where SppA localized in normoxia and hypoxia. Expression of the truncated SrbA‐N414 covering the SrbA sequence prior to the second transmembrane region rescued the growth of ΔdscA but not of ΔsppA in hypoxia. Unlike ΔdscA and ΔdscA;ΔsppA double mutants, in which SrbA cleavage was blocked, the molecular weight of cleaved SrbA increased in ΔsppA compared to the control strain in immunoblot analyses. Overall, our data demonstrate the sequential cleavage of SrbA by Dsc‐linked proteolysis followed by SppA, proposing a new model of RIP for SREBP cleavage in fungal hypoxia adaptation. Furthermore, the function of SppA in hypoxia adaptation was consistent in Aspergillus fumigatus, suggesting the potential roles of SppA in fungal pathogenesis.     

ALS/FTD‐associated FUS activates GSK‐3β to disrupt the VAPB–PTPIP51 interaction and ER–mitochondria associations

Defective FUS metabolism is strongly associated with amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD), but the mechanisms linking FUS to disease are not properly understood. However, many of the functions disrupted in ALS/FTD are regulated by signalling between the endoplasmic reticulum (ER) and mitochondria. This signalling is facilitated by close physical associations between the two organelles that are mediated by binding of the integral ER protein VAPB to the outer mitochondrial membrane protein PTPIP51, which act as molecular scaffolds to tether the two organelles. Here, we show that FUS disrupts the VAPB–PTPIP51 interaction and ER–mitochondria associations. These disruptions are accompanied by perturbation of Ca²⁺ uptake by mitochondria following its release from ER stores, which is a physiological read‐out of ER–mitochondria contacts. We also demonstrate that mitochondrial ATP production is impaired in FUS‐expressing cells; mitochondrial ATP production is linked to Ca²⁺ levels. Finally, we demonstrate that the FUS‐induced reductions to ER–mitochondria associations and are linked to activation of glycogen synthase kinase‐3β (GSK‐3β), a kinase already strongly associated with ALS/FTD.     

Murine response studies of insect cell (Sf9) expressed recombinant colorectal cancer vaccine candidate using surface plasmon resonance studies

The baculovirus vector expression system is considered a useful insect cell‐based platform for large‐scale production of recombinant biopharmaceutical glycoproteins. The GA733 antigenic protein is a 40 kDa cell surface glycoprotein that is overexpressed in human colorectal carcinoma. Three GA733‐expressing baculovirus vectors were constructed: (i) GA733 fused to the Fc fragment of human immunoglobulin G (GA733‐Fc); (ii) GA733‐Fc tagged with the endoplasmic reticulum (ER) retention signal KDEL (GA733‐FcK); and (iii) GA733‐FcK without the signal peptide [GA733‐FcK (w/o SP)]. These three recombinant proteins were expressed in Fall Armyworm (Spodoptera frugiperda) Sf9 insect cells. GA733‐Fc purified from insect culture medium (CM) and cell lysate (CL) (GA733^I‐Fc^CM and GA733^I‐Fc^CL, respectively), and GA733‐FcK and GA733‐FcK (w/o SP) purified from CL [GA733^I‐Fc^CL and GA733^I‐FcK^CL (w/o SP), respectively] were injected intraperitoneally into BALB/c mice [three times at 1 μg, with or without an adjuvant (aluminum hydroxide)]. Surface plasmon resonance (SPR) analysis (involving a chip with the immobilized GA733 antigen) showed that, in general, the serum samples from mice immunized with the adjuvant yielded stronger SPR signals than did those from mice immunized without an adjuvant. The serum samples from mice after the second injection yielded stronger signals than did those from mice before the second injection. The group immunized with GA733^I‐FcK^CL (glycosylated with an oligomannose) showed the strongest SPR signals. The SPR signals from the GA733^I‐FcK^CL group were stronger than or similar to those from mice immunized with mammalian‐cell‐expressed GA733‐Fc (GA733^M‐Fc). Thus, the baculovirus vector expression system can be used to produce immunogenic cancer glycoprotein.     

Comparative Analysis of Substrate-Free Cultured Oral Mucosal Epithelial Cell Sheets from Cells of Subjects with and without Stevens—Johnson Syndrome for Use in Ocular Surface Reconstruction

Purpose

To compare the regenerative potential of cultured oral mucosal epithelial cells sheets (COMECs) from Stevens-Johnson syndrome (SJS) subjects with those from non-SJS subjects.

Methods

Human oral mucosal epithelial cells from SJS and non-SJS subjects were cultured, and colony-forming efficiency (CFE), proliferative and migration potential, expression of cytokines/growth factors and stem cells were compared. COMECs from SJS and non-SJS subjects were transplanted into 12 limbal stem cell-deficient rabbits, and their regenerative potential was analyzed at 1 week after transplantation.

Results

CFE (p>0.05, student’s t test), cell proliferation potential (p>0.05, two-way ANOVA) and expression of the cytokeratins (K3, K4, K13, K19) in the oral mucosal epithelial cells from SJS subjects were similar to those of the cells from non-SJS subjects. The initial migratory potential of SJS cells was delayed compared to that of non-SJS cells (p <0.05, RM two-way ANOVA). The SJS cells expressed lower levels of EGF and higher levels of VEGF compared to that of non-SJS cells (p<0.05, one-way ANOVA). In vivo transplanted SJS-COMECs showed similar expression of K3, K4, and K13, proliferation markers (Ki-67; p>0.05, Mann-Whitney U test), and stem cell markers (p63; p>0.05, Mann-Whitney U test) compared to non-SJS COMECs. The initial epithelial defects in vivo were larger in the eyes treated with SJS-COMECs on day 3 (p<0.01, RM two-way ANOVA), but no differences were observed by day 7 between SJS- and non-SJS-COMECs.

Conclusions

These results suggest that, aside from differences in migratory potential, oral mucosal epithelial cells from SJS and non-SJS subjects are comparable in their regeneration potential in treating limbal stem cell deficiency.     

Lidocaine block of cardiac sodium channels

Lidocaine block of cardiac sodium channels was studied in voltage-clamped rabbit purkinje fibers at drug concentrations ranging from 1 mM down to effective antiarrhythmic doses (5-20 μM). Dose-response curves indicated that lidocaine blocks the channel by binding one-to-one, with a voltage-dependent K(d). The half-blocking concentration varied from more than 300 μM, at a negative holding potential where inactivation was completely removed, to approximately 10 μM, at a depolarized holding potential where inactivation was nearly complete. Lidocaine block showed prominent use dependence with trains of depolarizing pulses from a negative holding potential. During the interval between pulses, repriming of I (Na) displayed two exponential components, a normally recovering component (τless than 0.2 s), and a lidocaine-induced, slowly recovering fraction (τ approximately 1-2 s at pH 7.0). Raising the lidocaine concentration magnified the slowly recovering fraction without changing its time course; after a long depolarization, this fraction was one-half at approximately 10 μM lidocaine, just as expected if it corresponded to drug-bound, inactivated channels. At less than or equal to 20 μM lidocaine, the slowly recovering fraction grew exponentially to a steady level as the preceding depolarization was prolonged; the time course was the same for strong or weak depolarizations, that is, with or without significant activation of I(Na). This argues that use dependence at therapeutic levels reflects block of inactivated channels, rather than block of open channels. Overall, these results provide direct evidence for the “modulated-receptor hypothesis” of Hille (1977) and Hondeghem and Katzung (1977). Unlike tetrodotoxin, lidocaine shows similar interactions with Na channels of heart, nerve, and skeletal muscle.     

Comparison of phenological characteristics for several woody plants in urban climates

Phenological properties of woody species were compared between two urban climates during 1997 and 1998. The study areas were Chungdam Park, Chungdam-dong, Kangnam-gu, Seoul (the urban center, 43 species) and Namhan-sansung Area, Sansung-ri, Joongbu-myon, Kwangju Gun, Kyonggi Province (the urban periphery, 16 species). Distance between these sites was 13.5 km. The differences of budding, foliation, and flowering times (1997 versus 1998) were 10.9, 3.2, and 7.4 days, respectively. Species that budded and flowered earlier were strongly influenced by Nuttonson’s Index (Tn) of February and March, but those with later dates were only weakly influenced. Unlike for budding and flowering times, foliation time was determined by air temperature or other factors in the leaf-growing season rather than by Tn. The Tn influence over phenology was stronger in shrubs and lianas than in trees. Phenophases in Chungdam Park appeared earlier than those in the Namhansansung area. The phenological differences between the two areas were 7.3 days in budding time, 8.3 days in foliation time, and 10.2 days in flowering time in mean values, with variations among species. Based on flowering-time data, the phenological variation between the two areas was equivalent to a 2.5° latitude difference. Budding time varied the most (20 days) inZelkova serrate, compared with only 3 days forPrunus padus. Differences in foliation time ranged from 15 days (inAlnus hirsute andStyrax obassia) to 0 days (P. padus). Flowering time differences were largest (24 days) inRhododendron mucronulatum and smallest (2 days) inP. padus. One can conclude that heat pollution in the urban center in Seoul severely changed phenology, and that sensitivity to that pollution differed among plant species.