Comparative thermal adaptation in cicadas (Hemiptera: Cicadidae) inhabiting Mediterranean ecosystems

The thermal responses of cicadas inhabiting the Mediterranean ecosystems in Europe, North America, South Africa, and Australia are investigated. A total of 37 species and two subspecies from 17 genera representing eight tribes and three subfamilies of cicadas are investigated. The analysis includes species that are restricted to the Mediterranean ecosystem as well as those which also inhabit additional environments. The data suggest that cicadas adapt to the climate type regardless of particular types of plants within the various Mediterranean communities. Similarly, cicada thermal responses are independent of body size or taxonomic affinities. There is a wider range of body temperatures for the maximum voluntary tolerance temperature than for heat torpor or minimum flight temperatures. This diversity seems to be determined by the subdivision of the habitat used and the behavior of the species. All species possess relatively elevated heat torpor temperatures adapting to the general thermal characteristics of the Mediterranean ecosystem. The data suggest that cicadas adapt to the Mediterranean climate type regardless of the diversity of particular types of plants within the various communities, of body size or of taxonomic position.     

Separation of dissociated thyroid follicular and parfollicular cells: association of serotonin binding protein with parafollicular cells

Parafollicular cells (PC) of the sheep thyroid gland are neural crest derivatives that synthesize and release the biogenic amine serotonin (5-HT) as well as the hormone calcitonin. The thyroid also contains a highly specific serotonin-binding protein (SBP). Separation of dissociated thyroid cells was done to study the cellular localization of SBP and to develop a means of isolating PC for study. Various methods were used to obtain an enriched and purified population of PC. Minced thyroid glands were enzymatically dissociated and the cells were layered on a Ficoll linear density gradient. Fractions obtained from the gradient were examined for cell number, viability, 5-HT concentration, SBP activity, and morphology by electron microscopy. One of the fractions was found to be enriched in PC. High levels of 5-HT and SBP were also found in this fraction, whereas these levels were low where the majority of cells were found. This PC-rich fraction, however, contained numerous follicular cells (FC); therefore, additional approaches to cell separation were used. FC can be stimulated in vitro with thyroid stimulating hormone (TSH) to become intensely phagocytic. When stimulated cells were incubated in the presence of silica microspheres, the FC engulfed the microspheres, which were toxic to them. PC did not become phagocytic and were unharmed by the microspheres. Suspended cells, after incubation with microspheres, were centrifuged on a discontinuous gradient, and a PC-rich fraction was obtained. Silica, however, interfered with analysis of SBP. Another method to take advantage of the phagocytic potential of FC was therefore used. TSH-stimulated cell suspensions were passed through a column of sepharose to which thyroglobulin had been coupled. Stimulated FC apparently adhered to the beads and were retained by the columns. Fractions eluting from the columns were greatly enriched with PC. These fractions contained high levels of 5-HT and SBP, and considerably reduced FC contamination was found by quantitative electron microscopy. It is concluded that SBP is localized to PC in the sheep thyroid. The idea that these cells resemble serotonergic neurons in their mechanisms of 5-HT storage is supported.     

Carboxymethylated-κ-casein: A convenient tool for the identification of polyphenolic inhibitors of amyloid fibril formation

Reduced and carboxymethylated-κ-casein (RCM-κ-CN) is a milk-derived amyloidogenic protein that readily undergoes nucleation-dependent aggregation and amyloid fibril formation via a similar pathway to disease-specific amyloidogenic peptides like amyloid beta (Aβ), which is associated with Alzheimer’s disease. In this study, a series of flavonoids, many known to be inhibitors of Aβ fibril formation, were screened for their ability to inhibit RCM-κ-CN fibrilisation, and the results were compared with literature data on Aβ inhibition. Flavonoids that had a high degree of hydroxylation and molecular planarity gave good inhibition of RCM-κ-CN fibril formation. IC₅₀ values were between 10- and 200-fold higher with RCM-κ-CN than literature results for Aβ fibril inhibition, however, with few exceptions, they showed a similar trend in potency. The convenience and reproducibility of the RCM-κ-CN assay make it an economic alternative first screen for Aβ inhibitory activity, especially for use with large compound libraries.     

The statistics of identifying differentially expressed genes in Expresso and TM4: a comparison

Background  

Analysis of DNA microarray data takes as input spot intensity measurements from scanner software and returns differential expression of genes between two conditions, together with a statistical significance assessment. This process typically consists of two steps: data normalization and identification of differentially expressed genes through statistical analysis. The Expresso microarray experiment management system implements these steps with a two-stage, log-linear ANOVA mixed model technique, tailored to individual experimental designs. The complement of tools in TM4, on the other hand, is based on a number of preset design choices that limit its flexibility. In the TM4 microarray analysis suite, normalization, filter, and analysis methods form an analysis pipeline. TM4 computes integrated intensity values (IIV) from the average intensities and spot pixel counts returned by the scanner software as input to its normalization steps. By contrast, Expresso can use either IIV data or median intensity values (MIV). Here, we compare Expresso and TM4 analysis of two experiments and assess the results against qRT-PCR data.     

Echinococcus granulosus: variability of the host-protective EG95 vaccine antigen in G6 and G7 genotypic variants

Cystic hydatid disease in humans is caused by the zoonotic parasite Echinococcus granulosus. As an aid to control transmission of the parasite, a vaccine has been produced for prevention of infection in the parasite’s natural animal intermediate hosts. The vaccine utilizes the recombinant oncosphere protein, EG95. An investigation into the genetic variability of EG95 was undertaken in this study to assess potential antigenic variability in E. granulosus with respect to this host-protective protein. Gene-specific PCR conditions were first established to preferentially amplify the EG95 vaccine-encoding gene (designated eg95-1) from the E. granulosus genome that also contains several other EG95-related genes. The optimized PCR conditions were used to amplify eg95-1 from several parasite isolates in order to determine the protein-coding sequence of the gene. An identical eg95-1 gene was amplified from parasites showing a G1 or G2 genotype of E. granulosus. However, from isolates having a G6 or G7 genotype, a gene was amplified which had substantial nucleotide substitutions (encoding amino acid substitutions) compared with the eg95 gene family members. The amino acid substitutions of EG95 in the G6/G7 genotypes may affect the antigenicity/efficacy of the EG95 recombinant antigen against parasites of these genotypes. These findings indicate that characterization of eg95 gene family members in other strains/isolates of E. granulosus may provide valuable information about the potential for the EG95 hydatid vaccine to be effective against E. granulosus strains other than the G1 genotype.     

Twenty-four-hour variations in activity,core temperature,metabolic rate,and respiratory quotient in captive chinese pangolins

The circadian rhythms in activity, core temperature (T_c), O₂ consumption, CO₂ production, and respiratory quotient (RQ) were monitored in four captive Chinese pangolins (Manis pentadactyla). The pangolins were strictly nocturnal, never emerging from their nest before 1600 h, and their intermittent activity continued no later than 0230. As is usual in nocturnal mammals, the highest values observed in T_c, O₂ consumption, and CO₂ production occurred during the night; the lowest values occurred during the day. The magnitude of the variation in T_c, O₂ consumption, CO₂ production, and RQ averaged 1.2°C, 1.3 ml O₂ kg^?1 min^?1, 1.2 ml CO₂ kg^?1 min^?1, and 0.24, respectively. The circadian pattern in RQ was independent of activity, T_c, and the metabolic parameters and was of a different character than the patterns exhibited in the other variables. RQ remained constant at either a high or low level for long periods (8–10 h) and then increased or decreased relatively rapidly (1–2h) to the other level as in a square wave, whereas the rhythms in the other variables are similar to sine waves. The sharp increase in RQ was followed by a slow decline in T_c, and the sharp decline in RQ was followed by a slow increase in T_c.     

Induction of tumor cell apoptosis in vivo increases tumor antigen cross-presentation,cross-priming rather than cross-tolerizing host tumor-specific CD8 T cells

Cross-presentation of cell-bound Ags from established, solid tumors to CD8 cells is efficient and likely to have a role in determining host response to tumor. A number of investigators have predicted that when tumor Ags are derived from apoptotic cells either no response, due to Ag "sequestration," or CD8 cross-tolerance would ensue. Because the crucial issue of whether this happens in vivo has never been addressed, we induced apoptosis of established hemagglutinin (HA)-transfected AB1 tumors in BALB/c mice using the apoptosis-inducing reagent gemcitabine. This shrank the tumor by approximately 80%. This induction of apoptosis increased cross-presentation of HA to CD8 cells yet neither gross deletion nor functional tolerance of HA-specific CD8 cells were observed, based on tetramer analysis, proliferation of specific CD8 T cells, and in vivo CTL activity. Interestingly, apoptosis primed the host for a strong antitumor response to a second, virus-generated HA-specific signal in that administration of an HA-expressing virus after gemcitabine administration markedly decreased tumor growth compared with viral administration without gemcitabine. Thus tumor cell apoptosis in vivo neither sequesters tumor Ags nor cross-tolerizes tumor-specific CD8 cells. This observation has fundamental consequences for the development of tumor immunotherapy protocols and for understanding T cell reactivity to tumors and the in vivo immune responses to apoptotic cells.