Complete subunit sequences, structure and evolution of the 6 x 6-mer hemocyanin from the common house centipede, Scutigera coleoptrata.

Hemocyanins are large oligomeric copper-containing proteins that serve for the transport of oxygen in many arthropod species. While studied in detail in the Chelicerata and Crustacea, hemocyanins had long been considered unnecessary in the Myriapoda. Here we report the complete molecular structure of the hemocyanin from the common house centipede Scutigera coleoptrata (Myriapoda: Chilopoda), as deduced from 2D-gel electrophoresis, MALDI-TOF mass spectrometry, protein and cDNA sequencing, and homology modeling. This is the first myriapod hemocyanin to be fully sequenced, and allows the investigation of hemocyanin structure-function relationship and evolution. S. coleoptrata hemocyanin is a 6 x 6-mer composed of four distinct subunit types that occur in an approximate 2 : 2 : 1 : 1 ratio and are 49.5-55.5% identical. The cDNA of a fifth, highly diverged, putative hemocyanin was identified that is not included in the native 6 x 6-mer hemocyanin. Phylogenetic analyses show that myriapod hemocyanins are monophyletic, but at least three distinct subunit types evolved before the separation of the Chilopoda and Diplopoda more than 420 million years ago. In contrast to the situation in the Crustacea and Chelicerata, the substitution rates among the myriapod hemocyanin subunits are highly variable. Phylogenetic analyses do not support a common clade of Myriapoda and Hexapoda, whereas there is evidence in favor of monophyletic Mandibulata.     

The effect of light levels on daily patterns of chlorophyll fluorescence and organic acid accumulation in the tropical CAM treeClusia hilariana

Sandy plains are characteristic of the coastal region of Brazil. We investigated the diel patterns of changes in organic acid levels, leaf conductance and chlorophylla fluorescence for sun-exposed and shaded plants ofClusia hilariana, one of the dominant woody species in the sandy coastal plains of northern Rio de Janeiro state. Both exposed and shaded plants showed a typical CAM pattern with considerable diel oscillations in organic acid levels. The degradation of both malic and citric acids during the midday stomatal closure period could lead to potential CO₂ fixation rates of 28 mol m^-2 s^-1 in exposed leaves. Moreover, exposed leaves exhibited large increases in total non-photochemical quenching (q_N) accompanied by a substantial decrease in effective quantum yield during the course of the day. However, these potential high rates of CO₂ fixation and the increases inqn of exposed plants were not enough to maintain the primary electron acceptor of photosystem II (q_A) in a low reduction state, similar to that of shaded plants. As a result, there was a moderate increase in the reduction state of q_A throughout the day. Most of the decline in photochemical efficiency of exposed leaves ofC. hilariana was reversible, as evidenced by the high levels of pre-dawn potential quantum yields (F_v/F_m) and their rapid recovery after sunset. However, the depletion of the organic acid pool in the afternoon resulted in an accentuated subsequent drop in F_v/F_m, suggesting that prolonged periods of water stress accompanied by high irradiance levels may expose plants ofC. hilariana in unprotected habitats to the danger of photoinhibition.     

Localization of collagens and alkaline phosphatase activity during mineralization and ossification of human first rib cartilage

The localization of type X collagen and alkaline phosphatase activity was examined in order to gain a better understanding of tissue remodelling during development of human first rib cartilage. First rib cartilages from children and adolescents showed no staining for type X collagen and alkaline phosphatase activity. After onset of mineralization in the late second decade, a peripheral ossification process preceded by mineralized fibrocartilage could be distinguished from a more central one preceded by mineralized hyaline cartilage. No immunostaining for type X collagen was found in either type of cartilage. However, strong staining for alkaline phosphatase activity was detected around chondrocyte-like cells within fibrocartilage adjacent to the peripheral mineralization front, while a weaker staining pattern was observed around chondrocytes of hyaline cartilage near the central mineralization front. In addition, the territorial matrix of some chondrocytes within the hyaline cartilage revealed staining for type I collagen, suggesting that these cells undergo a dedifferentiation process, which leads to a switch from type II to type I collagen synthesis. The study provides evidence that mineralization of the hyaline cartilage areas in human first rib cartilage occurs in the absence of type X collagen synthesis but in the presence of alkaline phosphatase. Thus, mineralization of first rib cartilage seems to follow a different pattern from endochondral ossification in epiphyseal discs.     

Anamylopsoraceae — a new family of lichenized ascomycetes with stipitate apothecia (Lecanorales: Agyriineae)

The anatomy, chemistry and developmental morphology ofAnamylopsora pulcherrima is investigated. Some characters, including the ascus structure, suggest a close affinity with theAgyriaceae. However, the chemistry and the pycnidial structure differ as well as the ascoma ontogeny.Anamylopsora has a gymnocarpous ascoma development and the ascogonia are produced in stipes.Trapelia coarctata, as a typical member of theAgyriaceae, shows a hemiangiocarpous ascoma ontogeny. The anatomical, chemical and ontogenetical characters of several families are compared withAnamylopsora and it is shown that the genus is best placed in a monotypic familyAnamylopsoraceae Lumbsch & Lunke, fam. nova, which is placed in theAgyriineae (Lecanorales).This paper is dedicated to Prof. DrAino Henssen (Marburg) on the occasion of the 70th birthday.     

A simple statistical method for use in kinetic analysis based on Lineweaver-Burk plots

Statistical methods for the analysis of initial-velocity and/or inhibition data are described. They involve application of F tests (i) to determine goodness of fit to the first-order Michaelis-Menten equation, (ii) to predict the reaction mechanism by assessing slope and y-intercept effects in Lineweaver-Burk plots according to the inspection rules of Cleland [Cleland, W. W. (1963) Biochim. Biophys. Acta67, 188–196], (iii) to test the linearity of the replots of slopes or y-intercepts versus the reciprocal of the substrate concentration or the inhibitor concentration, and (iv) to estimate the true K_m or K_i values from these replots. The method serves to fill a gap in the kinetic analysis methodology between the antiquated graphical method and the sophisticated direct computer-fitting of data to a variety of possible rate equations. The entire theoretical and computational format is provided to allow the investigator to apply these statistical tests to his data using only a desk-top calculator.     

Der Einfluß von Objektart und früher Erfahrung auf die Diskriminationsleistungen von Haushühnern (Gallus domesticus)3

Main subject of the reported investigation is the question in which way the acquisition of a conditional discrimination is modified on the one hand by the kind of objects presented, on the other hand by early experience. 40 newly hatched chickens grew up either with all the wooden eggs or cubes used for training and test, or without these objects, or in a ‘natural’ (enriched) environment. The results prove an influence of the kind of objects — cubes are discriminated twice or three times as fast as wooden eggs — but no effects of the different environments on acquisition (number of trials for reaching learning criterion). Results are discussed under aspects of species-specific constraints on learning.     

Microphotometric analysis of NADH-tetrazolium reductase and alpha-glycerophosphate dehydrogenase in human quadriceps muscle.

Synopsis In serial cross-sections of human skeletal muscles stained for either NADH-tetrazolium reductase (NADH-TR) or -glycerophosphate dehydrogenase (-GPD), a linear relation was found between the total content of enzyme in a cell (expressed as the thickness of the section) and the absorbance of the formazan reaction product formed. Little variation (<4.8%) was found in the concentration of formazan (absorbance per unit thickness) when the same cell was measured in serial cross-sections of various thicknesses (2–10 m) along a longitudinal distance of at least 200 m along the cell. The reduction in enzyme activity was found to be negligible after aqueous preincubation. A maximum of 10–12% of the formazan produced in the NADH-TR reaction might be the result of nothing dehydrogenase activity, whereas this unspecific reaction might account for up to 20% of the formazan deposited in the -GPD reactions after 30 min incubation. The diffusion of Nitro BT into the tissue during the incubation period was found to be unhindered. The rates of formazan production decreased with increasing incubation time, especially in the -GPD reaction in both fibre types. The ratio of the mean absorbance of the formazan in type I fibres to that in type II fibres (in the same section) was 1.41 (coefficient of variation, 2.5%) in the NADH-TR reaction and 0.68 (coefficient of variation, 3.8%) in the -GPD reaction. These values were not affected either by variations in the incubation time (5–40 min) or by the thickness of the section (2–8 m). The concentrations of NADH-TR and -GPD seem to be constant along the length of the muscle fibre. The histochemical reactions reported, together with measurements of the thickness of the sections, seem suitable for the microphotometric quantification of the two enzymes in single fibres of human skeletal muscles.     

SOMA: A Single Oligonucleotide Mutagenesis and Cloning Approach

Modern biology research requires simple techniques for efficient and restriction site-independent modification of genetic material. Classical cloning and mutagenesis strategies are limited by their dependency on restriction sites and the use of complementary primer pairs. Here, we describe the Single Oligonucleotide Mutagenesis and Cloning Approach (SOMA) that is independent of restriction sites and only requires a single mutagenic oligonucleotide to modify a plasmid. We demonstrate the broad application spectrum of SOMA with three examples. First, we present a novel plasmid that in a standardized and rapid fashion can be used as a template for SOMA to generate GFP-reporters. We successfully use such a reporter to assess the in vivo knock-down quality of morpholinos in Xenopus laevis embryos. In a second example, we show how to use a SOMA-based protocol for restriction-site independent cloning to generate chimeric proteins by domain swapping between the two human hRMD5a and hRMD5b isoforms. Last, we show that SOMA simplifies the generation of randomized single-site mutagenized gene libraries. As an example we random-mutagenize a single codon affecting the catalytic activity of the yeast Ssy5 endoprotease and identify a spectrum of tolerated and non-tolerated substitutions. Thus, SOMA represents a highly efficient alternative to classical cloning and mutagenesis strategies.     

No support for the emergence of lichens prior to the evolution of vascular plants

The early‐successional status of lichens in modern terrestrial ecosystems, together with the role lichen‐mediated weathering plays in the carbon cycle, have contributed to the long and widely held assumption that lichens occupied early terrestrial ecosystems prior to the evolution of vascular plants and drove global change during this time. Their poor preservation potential and the classification of ambiguous fossils as lichens or other fungal–algal associations have further reinforced this view. As unambiguous fossil data are lacking to demonstrate the presence of lichens prior to vascular plants, we utilize an alternate approach to assess their historic presence in early terrestrial ecosystems. Here, we analyze new time‐calibrated phylogenies of ascomycete fungi and chlorophytan algae, that intensively sample lineages with lichen symbionts. Age estimates for several interacting clades show broad congruence and demonstrate that fungal origins of lichenization postdate the earliest tracheophytes. Coupled with the absence of unambiguous fossil data, our work finds no support for lichens having mediated global change during the Neoproterozoic‐early Paleozoic prior to vascular plants. We conclude by discussing our findings in the context of Neoproterozoic‐Paleozoic terrestrial ecosystem evolution and the paleoecological context in which vascular plants evolved.