Aspects of morphogenesis and function of diatom cell walls with implications for taxonomy

Summary Aspects of morphogenesis and morphology of diatom cell walls are reviewed to highlight functional correlations between wall structures and three-dimensional cytoplasmic activities during the cell cycle. Morphogenesis of the siliceous valve within the silica deposition vesicle is discussed in the light of the dependency on a precisely orchestrated moulding machinery, involving the cytoskeleton, mitochondria, endoplasmic reticulum, spacer vesicles produced by the Golgi apparatus, and the plasmalemma, in combination with adhesion of the cells to parts of the parental wall and localized plasmolyses. Sensitivity of morphogenetic events to fluctuations of external factors has implications for taxonomy.Abbreviations CF cleavage furrows - cPL cleavage plasmalemma - GB girdle bands - LP labiate process - LPA labiate process apparatus - MC microtubule center - mLP macro labiate process - MT microtubule - MTOC microtubules organizing center - PL plasmalemma - SDV silica deposition vesicle - SL SDV membrane - SpV spacer vesicles Dedicated to Professor Peter Sitte on the occasion of his 65th birthday     

Role of the Cys 2-Cys 10 disulfide bond for the structure, stability, and folding kinetics of ribonuclease T1.

The Cys 2-Cys 10 disulfide bond in ribonuclease T1 was broken by substituting Cys 2 and Cys 10 by Ser and Asn, respectively, as present in ribonuclease F1. This C2S/C10N variant resembles the wild-type protein in structure and in catalytic activity. Minor structural changes were observed by 2-dimensional NMR in the local environment of the substituted amino acids only. The thermodynamic stability of ribonuclease T1 is strongly reduced by breaking the Cys 2-Cys 10 bond, and the free energy of denaturation is decreased by about 10 kJ/mol. The folding mechanism is not affected, and the trans to cis isomerizations of Pro 39 and Pro 55 are still the rate-limiting steps of the folding process. The differences in the time courses of unfolding and refolding are correlated with the decrease in stability: the folding kinetics of the wild-type protein and the C2S/C10N variant become indistinguishable when they are compared under conditions of identical stability. Apparently, the Cys 2-Cys 10 disulfide bond is important for the stability but not for the folding mechanism of ribonuclease T1. The breaking of this bond has the same effect on stability and folding kinetics as adding 1 M guanidinium chloride to the wild-type protein.     

Molecular characterization of ura1, a mutant allele for orotidine-5'-monophosphate decarboxylase in Schizophyllum commune

Abstract The basis of the auxotrophic ural phenotype in Schizophyllum commune has been investigated. Two point mutations causing changes in conserved amino acid positions 62 (from lysine to glutamate) and 79 (from leucine to phenylalanine) most likely are the cause for the observed phenotype, whereas the overall gene structure was unchanged. Since reversion rates in this locus are extremely low, a single point mutation could not be expected to be the cause for the mutation. Besides the two point mutations expected to be induced by UV mutagenesis, the two alleles investigated from independently isolated strains differ by approximately 7% in nucleic acid sequence and about 3% in amino acid sequence, indicating a distant relationship between the strains used.     

Metabolism of 2,2'-dihydroxybiphenyl by Pseudomonas sp. strain HBP1: production and consumption of 2,2',3-trihydroxybiphenyl.

Cells of Pseudomonas sp. strain HBP1 grown on 2-hydroxy- or 2,2'-dihydroxybiphenyl contain NADH-dependent monooxygenase activity that hydroxylates 2,2'-dihydroxybiphenyl. The product of this reaction was identified as 2,2',3-trihydroxybiphenyl by 1H nuclear magnetic resonance and mass spectrometry. Furthermore, the monooxygenase activity also hydroxylates 2,2',3-trihydroxybiphenyl at the C-3' position, yielding 2,2',3,3'-tetrahydroxybiphenyl as a product. An estradiol ring cleavage dioxygenase activity that acts on both 2,2',3-tri- and 2,2',3,3'-tetrahydroxybiphenyl was partially purified. Both substrates yielded yellow meta-cleavage compounds that were identified as 2-hydroxy-6-(2-hydroxyphenyl)-6-oxo-2,4-hexadienoic acid and 2-hydroxy-6-(2,3-dihydroxyphenyl)-6-oxo-2,4-hexadienoic acid, respectively, by gas chromatography-mass spectrometry analysis of their respective trimethylsilyl derivatives. The meta-cleavage products were not stable in aqueous incubation mixtures but gave rise to their cyclization products, 3-(chroman-4-on-2-yl)pyruvate and 3-(8-hydroxychroman-4-on-2-yl)pyruvate, respectively. In contrast to the meta-cleavage compounds, which were turned over to salicylic acid and 2,3-dihydroxybenzoic acid, the cyclization products are not substrates to the meta-cleavage product hydrolase activity. NADH-dependent salicylate monooxygenase activity catalyzed the conversions of salicylic acid and 2,3-dihydroxybenzoic acid to catechol and pyrogallol, respectively. The partially purified estradiol ring cleavage dioxygenase activity that acted on the hydroxybiphenyls also produced 2-hydroxymuconic semialdehyde and 2-hydroxymuconic acid from catechol and pyrogallol, respectively.     

Evidence for two different blue-light-receptor systems for the fast responses of stimulation of photosynthetic capacity and acidification of the plant surface in brown algae

Two blue-light responses of Phaeophyta that are expressed within a few seconds of a blue-light stimulus were characterized with respect to their photoreception properties. The first response is the activation of red-light-saturated photosynthesis which can be stimulated to values up to 5 times the rates in red light, depending on the species. The second response is a blue-light-induced acidification measurable at the plant surface. Both responses have similar kinetic characteristics and thus led us initially to hypothesise that they were causally connected in the same transduction mechanism. The two responses have action spectra [measured for Ectocarpus siliculosus (Dillwyn) Lyngb. and Laminaria saccharina (L.) Lamouroux] that are indistinguishable within the relatively large limits of error. However, in all species tested, the threshold sensitivity for blue light of the photosynthetic response is lower than that of the pH-shift by a factor of 2 to 150. Furthermore, stimulation of photosynthesis is sensitive to the flavin inhibitors, KI and phenylacetic acid, but the pH response is not affected by these inhibitors. Thus, the blue-light-induced pH-shift does not cause the stimulation of photosynthesis. In contrast, the different fluence-response relationships of the two responses and particularly the differential effect of the inhibitors are clear evidence for the action of two independent transduction pathways and photoreceptor systems for blue light. At least photoreception for stimulation of photosynthesis involves a flavin-or and a pterin.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - PAA phenylacetic acid We thank Dr. C. A. Maggs for collecting P. pavonica. This research was supported by National Environment Research Council grant No. GR3/8102.     

Molecular characterization of the Salmonella typhimurium parE gene.

The DNA sequence of the wild type S. typhimurium parE gene was determined. The predicted protein has 96.7% amino acid identity with the ParE protein of E.coli, but is 29 amino acids longer, due to an additional basepair in the 3' end of the S. typhimurium gene. Subclones of the S. typhimurium parE gene localized the sites of four heat sensitive mutations within parE. The parE206 and parE374 mutations are identical (Val67-Met) and lie in a highly conserved region corresponding to the ATP binding pocket of GyrB. Two additional heat sensitive mutations were sequenced and predict the following amino acid substitutions: parE377 (Gly399-Ser) and parE493 (Thr583-Pro). All of the heat sensitive mutations lie in regions with strong amino acid homology to GyrB.     

The evolution and ecology of multiple antipredator defences

Prey seldom rely on a single type of antipredator defence, often using multiple defences to avoid predation. In many cases, selection in different contexts may favour the evolution of multiple defences in a prey. However, a prey may use multiple defences to protect itself during a single predator encounter. Such “defence portfolios” that defend prey against a single instance of predation are distributed across and within successive stages of the predation sequence (encounter, detection, identification, approach (attack), subjugation and consumption). We contend that at present, our understanding of defence portfolio evolution is incomplete, and seen from the fragmentary perspective of specific sensory systems (e.g., visual) or specific types of defences (especially aposematism). In this review, we aim to build a comprehensive framework for conceptualizing the evolution of multiple prey defences, beginning with hypotheses for the evolution of multiple defences in general, and defence portfolios in particular. We then examine idealized models of resource trade-offs and functional interactions between traits, along with evidence supporting them. We find that defence portfolios are constrained by resource allocation to other aspects of life history, as well as functional incompatibilities between different defences. We also find that selection is likely to favour combinations of defences that have synergistic effects on predator behaviour and prey survival. Next, we examine specific aspects of prey ecology, genetics and development, and predator cognition that modify the predictions of current hypotheses or introduce competing hypotheses. We outline schema for gathering data on the distribution of prey defences across species and geography, determining how multiple defences are produced, and testing the proximate mechanisms by which multiple prey defences impact predator behaviour. Adopting these approaches will strengthen our understanding of multiple defensive strategies.     

Recruitment of epidermal growth factor and transferrin receptors into coated pits in vitro: differing biochemical requirements.

The biochemical requirements for epidermal growth factor (EGF) and transferrin receptor-mediated endocytosis were compared using perforated human A431 cells. Morphological studies showed that horseradish peroxidase (HRP)-conjugated EGF and gold-labeled antitransferrin (Tfn) receptor antibodies were colocalized during endocytosis in vitro. The sequestration of both ligands into deeply invaginated coated pits required ATP hydrolysis and cytosolic factors and was inhibited by GTP gamma S, indicating mechanistic similarities. Importantly, several differences in the biochemical requirements for sequestration of EGF and Tfn were also detected. These included differing requirements for soluble AP (clathrin assembly protein) complexes, differing cytosolic requirements, and differing sensitivities to the tyrosine kinase inhibitor, genistein. The biochemical differences detected between EGF and Tfn sequestration most likely reflect specific requirements for the recruitment of EGF-receptors (R) into coated pits. This assay provides a novel means to identify the molecular bases for these biochemical distinctions and to elucidate the mechanisms involved in ligand-induced recruitment of EGF-R into coated pits.     

Monoclonal antibody uptake in B-cell lymphomas: Experimental studies in nude mouse xenografts

Accumulation of radiolabelled monoclonal antibodies (mAb) in human B-lymphoma xenografts was found to result in two distinct patterns. The basic elements leading to these patterns were elucidated by autoradiographic and immunohistological analysis applied to the nude mouse xenografts BJAB and OCI.LY1. With BJAB, accumulation occurred exclusively in peripheral cell layers of the lymphoma nodule, while central areas were not accessible irrespective of mAb dose. This feature was the consequence of an inefficient transport across intratumoral vessels together with peripheral mAb supply through a subcapsular pseudosinus. With OCI.LY1, intratumoral vessels showed generalized leakiness. Furthermore, interstitial transport was operative to a fair extent, such that in early images multiple sites of mAb extravasation were obvious, which coalesced during the course of prolonged uptake. The pattern of peripheral mAb uptake resulted in a low overall tumour uptake, while multifocal uptake yielded substantial accumulation values.