ONTOGENETIC NICHE SHIFT IN THE BRACHIOPOD TEREBRATALIA TRANSVERSA: RELATIONSHIP BETWEEN THE LOSS OF ROTATION ABILITY AND ALLOMETRIC GROWTH

Abstract: Many articulated brachiopods experience marked life habit variations during ontogeny because they experience their fluid environment at successively higher Reynolds numbers, and they can change the configuration of their inhalant and exhalant flows as body size increases. We show that the extant brachiopod Terebratalia transversa undergoes a substantial ontogenetic change in reorientation governed by rotation around the pedicle. T. transversa′s reorientation angle (maximum ability to rotate on the pedicle) decreases during ontogeny, from 180 degrees in juveniles to 10–20 degrees in individuals exceeding 5 mm, to complete cessation of rotation in individuals larger than 10 mm. Rotation ability is substantially reduced after T. transversa achieves the adult lophophore configuration and preferred orientation with respect to ambient water currents at a length of 2.5–5 mm. We hypothesize that the rotation angle of T. transversa is determined mainly by the position of ventral and dorsal points of attachment of dorsal pedicle muscles relative to the pedicle. T. transversa shows a close correlation between the ontogenetic change in reorientation angle and ontogeny of morphological traits that are related to points of attachment of dorsal pedicle muscles, although other morphological features can also limit rotation in the adult stage. The major morphological change in cardinalia shape and the observed reduction of rotation affect individuals 2.5–10 mm in length. The position of ventral insertions of dorsal pedicle muscles remains constant, but contraction of dorsal pedicle muscles is functionally handicapped because dorsal insertions shift away from the valve midline, rise above the dorsal valve floor, and become limited by a wide cardinal process early in ontogeny (<5 mm). The rate of increase of cardinal process width and of distance between dorsal pedicle muscle scars substantially decreases in the subadult stage (5–10 mm), and most of the cardinalia shell traits grow nearly isometrically in the adult stage (>10 mm). T. transversa attains smaller shell length in crevices than on exposed substrates. The proportion of small‐sized individuals and population density is lower on exposed substrates than in crevices, indicating higher juvenile mortality on substrates prone to grazing and physical disturbance. The loss of reorientation ability can be a consequence of morphological changes that strengthen substrate attachment and maximize protection against biotic or physical disturbance (1) by minimizing torques around the pedicle axis and/or (2) by shifting energy investments into attachment strength at the expense of the cost involved in reorientation.     

Gene silencing and virus resistance based on defective interfering constructs in transgenic Nicotiana benthamiana is not linked to accumulation of siRNA

RNA interference (RNAi) was established in Nicotiana benthamiana plants by introducing constructs containing a defective interfering (DI) sequence from Tomato bushy stunt virus (TBSV) in combination with a conserved sense-sequence from the target Grapevine fanleaf virus (GFLV). Silencing in plants was confirmed by Agrobacterium-mediated infiltration of a GFP-sensor containing the GFLV-derived target sequence. The transgene-induced RNAi led to silencing of the GFP-sensor and GFP fluorescence was absent post-infiltration. In plants without GFP fluorescence after infiltration with the GFP-sensor, siRNA specific to GFP and the target virus sequence could not be detected. In contrast, infiltrated leaves of wild type and transgenic plants showing GFP fluorescence after infiltration revealed accumulation of siRNA specific to the sequence of the sensor. Silencing could be inhibited by co-infiltration using a p19 silencing suppressor construct together with the GFP-sensor, which always resulted in bright GFP fluorescence. In parallel, virus resistance of transgenic Nicotiana benthamiana was investigated via challenge inoculation with GFLV. Our results indicate that efficient RNAi in transgenic plants does not necessarily lead to a detectable accumulation of siRNA.     

Heterogeneity of phosphorus distribution in a patterned landscape,the Florida Everglades

The biologically mediated transfer of nutrients from one part of a landscape to another may create nutrient gradients or subsidize the productivity at specific locations. If limited, this focused redistribution of the nutrient may create non-random landscape patterns that are unrelated to underlying environmental gradients. The Florida Everglades, USA, is a large freshwater wetland that is patterned with tree islands, elevated areas that support woody vegetation. A survey of 12 tree islands found total soil phosphorus levels 3–114 times greater on the island head than the surrounding marsh, indicating that the Florida Everglades is not a homogeneous oligotrophic system. It was estimated that historically 67% of the phosphorus entering the central Everglades was sequestered on tree islands, which are ~3.8% of the total land area. This internal redistribution of phosphorus onto tree islands due to the establishment of trees may be one reason that marshes have remained oligotrophic and may explain the spatial differentiation of the patterned Everglades landscape.     

Experimental silicification of the extremophilic Archaea Pyrococcus abyssi and Methanocaldococcus jannaschii: applications in the search for evidence of life in early Earth and extraterrestrial rocks

Hydrothermal activity was common on the early Earth and associated micro‐organisms would most likely have included thermophilic to hyperthermophilic species. 3.5–3.3 billion‐year‐old, hydrothermally influenced rocks contain silicified microbial mats and colonies that must have been bathed in warm to hot hydrothermal emanations. Could they represent thermophilic or hyperthermophilic micro‐organisms and if so, how were they preserved? We present the results of an experiment to silicify anaerobic, hyperthermophilic micro‐organisms from the Archaea Domain Pyrococcus abyssi and Methanocaldococcus jannaschii, that could have lived on the early Earth. The micro‐organisms were placed in a silica‐saturated medium for periods up to 1 year. Pyrococcus abyssi cells were fossilized but the M. jannaschii cells lysed naturally after the exponential growth phase, apart from a few cells and cell remains, and were not silicified although their extracellular polymeric substances were. In this first simulated fossilization of archaeal strains, our results suggest that differences between species have a strong influence on the potential for different micro‐organisms to be preserved by fossilization and that those found in the fossil record represent probably only a part of the original diversity. Our results have important consequences for biosignatures in hydrothermal or hydrothermally influenced deposits on Earth, as well as on early Mars, as environmental conditions were similar on the young terrestrial planets and traces of early Martian life may have been similarly preserved as silicified microfossils.     

Oligoproline effects on polyglutamine conformation and aggregation

There are nine known expanded CAG repeat neurological diseases, including Huntington's disease (HD), each involving the repeat expansion of polyglutamine (polyGln) in a different protein. Similar conditions can be induced in animal models by expression of the polyGln sequence alone or in other protein contexts. Besides the polyGln sequence, the cellular context of the disease protein, and the sequence context of the polyGln within the disease protein, are both likely to contribute to polyGln physical behavior and to pathology. In HD, the N-terminal, exon-1 segment of the protein huntingtin contains the polyGln sequence immediately followed by an oligoproline region. We show here that introduction of a P10 sequence C-terminal to polyGln in synthetic peptides decreases both the rate of formation and the apparent stability of the amyloid-like aggregates associated with this family of diseases. The sequence can be trimmed to P6 without altering the suppression, but a P3 sequence is ineffective. Spacers up to at least three amino acid residues in length can be inserted between polyGln and P10 without altering this effect. There is no suppression, however, when the P10 sequence is either placed on the N-terminal side of polyGln or attached to polyGln via a side-chain tether. The nucleation mechanism of a Q40 sequence is unchanged upon addition of a P10 C-terminal extension, yielding a critical nucleus of one. The effects of oligoPro length and structural context on polyGln aggregation are correlated strongly with alterations in the circular dichroism spectra of the monomeric peptides. For example, the P10 sequence eliminates the small amount of alpha helical content otherwise exhibited by the Q40 sequence. The P10 sequence may suppress aggregation by stabilizing an aggregation-incompetent conformation of the monomer. The effect is transportable: a P10 sequence fixed to the C terminus of the sequence Abeta similarly modulates amyloid fibril formation.     

A mutation in aminopeptidase N (CD13) isolated from a patient suffering from leukemia leads to an arrest in the endoplasmic reticulum

Human aminopeptidase N (APN) is used as a routine marker for myelomonocytic cells in hematopoietic malignant disorders. Its gene and surface expressions are increased in cases of malignant transformation, inflammation, or T cell activation, whereas normal B and resting T cells lack detectable APN protein expression. In this study we elucidated the intracellular distribution, expression pattern, and enzymatic activity of a naturally occurring mutation in the coding region of the APN gene. At physiological temperatures the mutant protein is enzymatically inactive, persists as a mannose-rich polypeptide in the endoplasmic reticulum, and is ultimately degraded by an endoplasmic reticulum-associated degradation pathway. It shows in part the distinct behavior of a temperature-sensitive mutant with a permissive temperature of 32 degrees C, leading to correct sorting of the Golgi compartment accompanied by the acquisition of proper glycosylation but without reaching the cell-surface membrane and without regaining its enzymatic activity. Because the patient bearing this mutation suffered from leukemia, possible links to the pathogenesis of leukemia are discussed.     

Scanning cysteine mutagenesis analysis of Abeta-(1-40) amyloid fibrils

We describe here the use of cysteine substitution mutants in the Alzheimer disease amyloid plaque peptide Abeta-(1-40) to probe amyloid fibril structure and stabilization. In one approach, amyloid fibrils were grown from Cys mutant peptides under reducing conditions and then challenged with an alkylating agent to probe solvent accessibility of different residues in the fibril. In another approach, monomeric Cys mutants, either in the thiol form or modified with iodoacetic acid or methyl iodide, were grown into amyloid fibrils, and the equilibrium position at the end of the amyloid formation reaction was quantified by determining the concentration of monomeric Abeta. The DeltaG values of fibril elongation obtained were then compared in order to provide information on the environment of each residue side chain in the fibril. In general, Cys residues in the N and C termini of Abeta-(1-40) were not only accessible to alkylation in the fibril state but also, when modified in the monomeric state, did not greatly impact fibril stability; these observations were consistent with previous indications that these portions of the peptide are not part of the amyloid core. In contrast, residues 16-19 and 31-34 were not only uniformly inaccessible to alkylation in the fibril state, but their modification with the negatively charged carboxymethyl group in monomeric Abeta also destabilized fibril elongation, confirming other data showing that these segments are likely packed into a hydrophobic amyloid core. Residues 20, 30, and 35, flanking these implicated beta-sandwich regions, are accessible to alkylation in the fibril indicating a location in solvent exposed structure.     

T tubules and surface membranes provide equally effective pathways of carbonic anhydrase-facilitated lactic acid transport in skeletal muscle

We have studied lactic acid transport in the fast mouse extensor digitorum longus muscles (EDL) by intracellular and cell surface pH microelectrodes. The role of membrane-bound carbonic anhydrases (CA) of EDL in lactic acid transport was investigated by measuring lactate flux in muscles from wildtype, CAIV-, CAIX- and CAXIV-single ko, CAIV-CAXIV double ko and CAIV-CAIX-CAXIV-triple ko mice. This was complemented by immunocytochemical studies of the subcellular localization of CAIV, CAIX and CAXIV in mouse EDL. We find that CAXIV and CAIX single ko EDL exhibit markedly but not maximally reduced lactate fluxes, whereas triple ko and double ko EDL show maximal or near-maximal inhibition of CA-dependent lactate flux. Interpretation of the flux measurements in the light of the immunocytochemical results leads to the following conclusions. CAXIV, which is homogeneously distributed across the surface membrane of EDL fibers, facilitates lactic acid transport across this membrane. CAIX, which is associated only with T tubular membranes, facilitates lactic acid transport across the T tubule membrane. The removal of lactic acid from the lumen of T tubuli towards the interstitial space involves a CO2-HCO3- diffusional shuttle that is maintained cooperatively by CAIX within the T tubule and, besides CAXIV, by the CAIV, which is strategically located at the opening of the T tubules. The data suggest that about half the CA-dependent muscular lactate flux occurs across the surface membrane, while the other half occurs across the membranes of the T tubuli.