Purification, characterization and stability of barley grain peroxidase BP 1, a new type of plant peroxidase

The major peroxidase of barley grain (BP 1) has enzymatic and spectroscopic properties that are very differeant from those of other known plant peroxidases (EC 1.11.1.7) and can therefore contribute to the understanding of the many physiological functions ascribed to these enzymes. To study the structure-function relationships of this unique model peroxidase, large-scale and Jaboratory-scale purifications have been developed. The two batches of pure BP 1 obtained were identical in their enzymatic and spectral properties, and confirmed that BP 1 is different from the prototypical horseradish peroxidase isoenzyme C (HRP C). However, when measuring the specific activity of BP 1 at pH 4.0 in the presence of 1 m M C_aCl₂, the enzyme was as competent as HRP C at neutral pH towards a variety of substrates (m M mg⁻¹ min⁻¹): coniferyl alcohol (930±48), caffeic acid (795±53), ABTS (2,2'-azino-di-[3-ethyl-benzothiazoline-(6)-sulfonic acid]) (840±47), ferulic acid (415±20), p -coumaric acid (325±12), and guaiacol (58±3). The absorption spectrum of BP 1 is blue-shifted compared to that of HRP C with a Soret maximum of 399–402 nm, depending on pH. The prosthetic group was shown to be iron-protoporphyrin IX, which is characteristic of plant peroxidases. BP 1 is stable from pH 3 to 11, indicating that its unusual spectral characteristics do not result from enzyme instability. The thermostability is also normal with a melting temperature of 75°C at pH 6.6, and 67°C at pH 4.0 and 8.3. It is clear that the unusual properties of BP 1 are genuine, and reflect a novel regulation of plant peroxidase function.     

Ultrastructure of snail grazing damage to calcicolous lichens

The identification of damaged lichens is often difficult due to changes in the morphology of regenerating specimens. We examined the Ultrastructure of grazing damages to four species of calcicolous lichens ( Aspicilia calcarea, Physcia adscendens, Tephromela atra and Xanthoria parietina ) and free-living cyanobacteria (family Chroococcaceae) caused by individuals of four species of land snails ( Chondrina clienta, Balea perversa, Clausilia bidentata and Helicigona lapicida ). We also investigated the radular structure of the four lichen-feeding snails to examine whether differences in radular morphology result in species-specific grazing damages. Individuals of all four snail species removed the cyanobacteria layer covering the limestone or lichen surfaces. The four lichen species were grazed to a different extent by the different snail species. SEM-images showed that B. perversa left distinct depressions on the thalli of A. calcarea , whereas H. lapicida grazed off the thalli of this lichen rather evenly. Both snail species left visible radular traces on the lichen surface. In contrast, Ch. clienta left shallow depressions without radular traces on the thalli of A. calcarea. In Tephromela atra , grazing damages were observed on both thallus and ascocarp. Ascocarps of T. atra were partly grazed by B. perversa. Helicigona lapicida grazing on T. atra removed more or less evenly the entire lichen tissue including the ascocarps. In foliose lichens, grazing by Ch. clienta, B. perversa and Cl. bidentata resulted in depressions of different depths, while H. lapicida removed entire pieces of the thalli. In general, radular traces were less distinct in foliose lichens than in crustose lichens.     

Effects of digestive status on the reptilian gut

Reptiles, including the Burmese python, Python molurus bivittatus, that feed at infrequent intervals show a prominent increase in gastrointestinal mass, metabolism and brush border transport rates after feeding. Current knowledge and theories around these phenomena, as well as studies on the innervation of the reptilian gut, are summarised in this review. Little is known about the putative changes in the nervous and humoral control systems of the gut, and it is not known whether feeding affects innervation and motility of the stomach and intestine. Using immunohistochemistry, we have investigated possible up/down regulation of several neurotransmitters in specimens that had been fasted for a minimum of 3 weeks and specimens that had ingested a large meal 2 days before the experiments were conducted. There were no major changes in the innervation by nerves containing calcitonin gene-related peptide (CGRP), galanin, nitric oxide synthase (NOS), pituitary adenylate cyclase-activating polypeptide (PACAP), somatostatin (SOM), substance P/neurokinin A (SP/NKA), or vasoactive intestinal polypeptide (VIP)-like immunoreactivity. Nor did we find any differences in the effect of substance P (stomach and intestine), galanin (intestine), or bradykinin (intestine) on motility in strip preparations from the gut wall. A significant increase in dry weight of the intestine was obtained 48 h after feeding. We conclude that although there are considerable changes in gut thickness and absorptive properties after feeding, the smooth muscle and its control appear little affected.     

Women's Greater Ability to Perceive Happy Facial Emotion Automatically: Gender Differences in Affective Priming

There is evidence that women are better in recognizing their own and others' emotions. The female advantage in emotion recognition becomes even more apparent under conditions of rapid stimulus presentation. Affective priming paradigms have been developed to examine empirically whether facial emotion stimuli presented outside of conscious awareness color our impressions. It was observed that masked emotional facial expression has an affect congruent influence on subsequent judgments of neutral stimuli. The aim of the present study was to examine the effect of gender on affective priming based on negative and positive facial expression. In our priming experiment sad, happy, neutral, or no facial expression was briefly presented (for 33 ms) and masked by neutral faces which had to be evaluated. 81 young healthy volunteers (53 women) participated in the study. Subjects had no subjective awareness of emotional primes. Women did not differ from men with regard to age, education, intelligence, trait anxiety, or depressivity. In the whole sample, happy but not sad facial expression elicited valence congruent affective priming. Between-group analyses revealed that women manifested greater affective priming due to happy faces than men. Women seem to have a greater ability to perceive and respond to positive facial emotion at an automatic processing level compared to men. High perceptual sensitivity to minimal social-affective signals may contribute to women's advantage in understanding other persons' emotional states.     

Green fluorescent protein as a convenient and versatile marker for studies on functional genomics in Drosophila

Gene function can be deduced from lack or gain of activity. For the manipulation of gene doses or activity in Drosophila, a set of P-based vectors was constructed containing green fluorescent protein as marker. pBLUEi, pGREENi and pYELLi were designed for large insert transformation. Mosaicism was generated in vivo with pFlipG which is also ideal for targeted gene disruption. Tissue-specific gene silencing in vivo was performed with the vector set pHIBS and pUdsGFP. pHIBS allows easy cloning and shuttling of double-headed constructs. With pUdsGFP, double stranded RNA can be produced in defined patterns and the area of interference simultaneously visualized by green fluorescence. We demonstrate nearly complete silencing of a ubiquitously expressed gene in a tissue-specific manner.     

A molecular phylogeny of the African widowbirds and bishops, Euplectes spp. (Aves: Passeridae: Ploceinae)

The elaborate male displays and plumage ornaments in the African widowbirds and bishops (Euplectes spp.) have inspired classic studies on mating systems and sexual selection. In order to study the extreme divergence in ornament design and expression in this group, we present and discuss a well-supported molecular phylogeny of the genus and its placement within the Ploceinae subfamily. Parsimony and Bayesian analyses were performed on 2557bp of mitochondrial DNA (ATP6, Cyt b, ND2 and ND3) and a nuclear intron (G3PDH). All 17 Euplectes species, and 31 of 51 suggested subspecies, were included, as well as eight Ploceinae outgroups from four genera (Amblyospiza, Ploceus, Quelea and Foudia). Our results show monophyly of Euplectes, but not of the intrageneric groupings of bishops and widowbirds. Most notably, the Red-collared Widowbird E. ardens belongs to a subclade of bishops, and not to the sister subclade of 'true' widowbirds. Furthermore, the two bishops E. afer and E. aureus represent lineages that branched off before this basal split, which also refutes the proposed superspecies of E. afer and E. diadematus. Also somewhat surprisingly, and despite the striking plumage similarities among the red bishops, E. franciscanus is not closely related to either E. nigroventris or E. orix (of which it until recently was considered a subspecies). Finally, the Mountain Marsh Widowbird E. psammocromius is likely closest to the Long-tailed Widowbird E. progne, and not, as previously thought, to the Marsh Widowbird E. hartlaubi.     

Photodynamic targeting of EGFR does not predict the treatment outcome in combination with the EGFR tyrosine kinase inhibitor Tyrphostin AG1478

Photodynamic therapy (PDT) is a selective treatment modality against cancer. PDT is based on the preferential retention of photosensitizers (PSs), in the tumour and subsequent light exposure which activates the PS and generates reactive oxygen species. Multimodality therapy is increasingly relevant in cancer treatment and PDT has been shown as an effective adjuvant to other anti-cancer modalities. The present study reports on the combination of PDT and an epidermal growth factor receptor (EGFR) specific tyrosine kinase inhibitor (TKI), Tyrphostin AG1478. The combination was studied in two cell lines; A-431 and NuTu-19, expressing EGFR and sensitive to Tyrphostin treatment, but with different sensitivity towards photochemical EGFR damage. A-431 cells were treated with the PS meso-tetraphenylporphine with 2 sulfonate groups on adjacent phenyl rings (TPPS(2a)) in order to target mainly the endo/lysosomal compartments (18 h incubation followed by a 4 h chase in drug-free medium) or the plasma membrane (30 min incubation) upon light exposure. The EGFR was inhibited after PDT in A-431 cells only when TPPS(2a) was located on the plasma membrane, but both treatment regimes resulted in synergistic inhibition of cell growth when combined with Tyrphostin. TPPS(2a) treatment of NuTu-19 cells, designed for endo/lysosomal localization, followed by light attenuated EGFR phosphorylation but resulted in additive or antagonistic effects on cell growth when Tyrphostin was administered prior to or after PDT respectively. It was therefore concluded that photochemical damage of EGFR does not predict the treatment outcome when PDT is combined with Tyrphostin.