Molecular cloning and expression of the cDNA for a novel A2-adenosine receptor subtype.

A novel adenosine receptor subtype has been cloned from a rat brain cDNA library using a probe generated by the polymerase chain reaction. The cDNA, designated RFL9, encodes a protein of 332 amino acids. The structure of RFL9 is most similar to that of the recently cloned rat A2-adenosine receptor, with a sequence identity of 73% within the presumed seven transmembrane domains. Expression of RFL9 in COS-6M cells resulted in ligand binding and functional activity characteristics of an adenosine receptor that is coupled positively to adenylyl cyclase. Examination of the tissue distribution of RFL9 mRNA by Northern blot analysis showed a restricted distribution with highest levels expressed in large intestine, cecum, and urinary bladder; this pattern was distinct from that of either the A1- or A2-adenosine receptor mRNAs. In situ hybridization studies of RFL9 mRNA showed no specific hybridization pattern in brain, but a hybridization signal was readily observed in the hypophyseal pars tuberalis. Thus, RFL9 encodes a novel A2-adenosine receptor subtype.     

Characterization of Dinophysis spp. (Dinophyceae,Dinophysiales) from the mid-Atlantic region of the United States1

Due to the increasing prevalence of Dinophysis spp. and their toxins on every US coast in recent years, the need to identify and monitor for problematic Dinophysis populations has become apparent. Here, we present morphological analyses, using light and scanning electron microscopy, and rDNA sequence analysis, using a ~2-kb sequence of ribosomal ITS1, 5.8S, ITS2, and LSU DNA, of Dinophysis collected in mid-Atlantic estuarine and coastal waters from Virginia to New Jersey to better characterize local populations. In addition, we analyzed for diarrhetic shellfish poisoning (DSP) toxins in water and shellfish samples collected during blooms using liquid-chromatography tandem mass spectrometry and an in vitro protein phosphatase inhibition assay and compared this data to a toxin profile generated from a mid-Atlantic Dinophysis culture. Three distinct morphospecies were documented in mid-Atlantic surface waters: D. acuminata, D. norvegica, and a “small Dinophysis sp.” that was morphologically distinct based on multivariate analysis of morphometric data but was genetically consistent with D. acuminata. While mid-Atlantic D. acuminata could not be distinguished from the other species in the D. acuminata-complex (D. ovum from the Gulf of Mexico and D. sacculus from the western Mediterranean Sea) using the molecular markers chosen, it could be distinguished based on morphometrics. Okadaic acid, dinophysistoxin 1, and pectenotoxin 2 were found in filtered water and shellfish samples during Dinophysis blooms in the mid-Atlantic region, as well as in a locally isolated D. acuminata culture. However, DSP toxins exceeded regulatory guidance concentrations only a few times during the study period and only in noncommercial shellfish samples.     

Protocol of a prospective study on the diagnostic value of transcranial duplex scanning of the substantia nigra in patients with parkinsonian symptoms

Background  

Parkinson's disease (PD) is the second most common neurodegenerative disorder. As there is no definitive diagnostic test, its diagnosis is based on clinical criteria. Recently transcranial duplex scanning (TCD) of the substantia nigra in the brainstem has been proposed as an instrument to diagnose PD. We and others have found that TCD scanning of substantia nigra duplex is a relatively accurate diagnostic instrument in patients with parkinsonian symptoms. However, all studies on TCD so far have involved well-defined, later-stage PD patients, which will obviously lead to an overestimate of the diagnostic accuracy of TCD.     

Proteomic traces of speciation

Recent work has shown that the network of structural similarity between protein domains exhibits a power-law distribution of edges per node. The scale-free nature of this graph, termed the protein domain universe graph or PDUG, may be reproduced via a divergent model of structural evolution. The performance of this model, however, does not preclude the existence of a successful convergent model. To further resolve the issue of protein structural evolution, we explore the predictions of both convergent and divergent models directly. We show that when nodes from the PDUG are partitioned into subgraphs on the basis of their occurrence in the proteomes of particular organisms, these subgraphs exhibit a scale-free nature as well. We explore a simple convergent model of structural evolution and find that the implications of this model are inconsistent with features of these organismal subgraphs. Importantly, we find that biased convergent models are inconsistent with our data. We find that when speciation mechanisms are added to a simple divergent model, subgraphs similar to the organismal subgraphs are produced, demonstrating that dynamic models can easily explain the distributions of structural similarity that exist within proteomes. We show that speciation events must be included in a divergent model of structural evolution to account for the non-random overlap of structural proteomes. These findings have implications for the long-standing debate over convergent and divergent models of protein structural evolution, and for the study of the evolution of organisms as a whole.     

Toxic activity from cultures of Karlodinium micrum (=Gyrodinium galatheanum) (Dinophyceae)—a dinoflagellate associated with fish mortalities in an estuarine aquaculture facility

The goal of this study was to test for, and partially characterize, toxic activity associated with the dinoflagellate Karlodinium micrum. Since 1996, three fish kill events associated with blooms of K. micrum have occurred at HyRock Fish Farm, an estuarine pond aquaculture facility raising hybrid striped bass on the Chesapeake Bay, MD, USA. Using an assay based on the lysis of rainbow trout erythrocytes, cultures of a Chesapeake Bay isolate of K. micrum have been shown to produce toxic substances which are released upon cell disturbance or damage. The LC₅₀ for hemolysis of a sonicated cell suspension was 2.4×10⁴ cells ml⁻¹, well within the range of cell concentrations observed associated with fish kills. The toxic activity from K. micrum cells and culture filtrates was traced to two distinct fractions that co-elute with polar lipids. The LC₅₀ for hemolysis of the larger of these two fractions (Tox A) was 284 ng ml⁻¹ while the LC₅₀ of the second, smaller, fraction (Tox B) was 600 ng ml⁻¹. For comparison, the LC₅₀ for the standard hemolysin saponin was 3203 ng ml⁻¹. At concentrations of 800 and 2000 ng ml⁻¹, respectively, Tox A was further shown to be ichthyotoxic to zebrafish (Danio rerio) larvae (80% mortality), and cytotoxic to a mammalian GH(4)C(1) cell line (100% LDH release). At a concentration of 600 ng ml⁻¹ Tox B was shown to be cytotoxic to a mammalian GH(4)C(1) cell line (>30% LDH release), but not ichthyotoxic to zebrafish (D. rerio) larvae up to a concentration of 250 ng ml⁻¹. Although treatment with either algicidal copper or potassium permanganate caused significant lysis of K. micrum cells (>70%), toxic activity was released after treatment with copper and eliminated following treatment with potassium permanganate. This observation in cultures is consistent with observations made at HyRock Fish Farm where significantly higher mortality was observed following treatment of a K. micrum bloom with copper sulfate compared to treatment with potassium permanganate. This study represents the first direct evidence of the toxicity of K. micrum isolated from the Chesapeake Bay.     

Floristic variation and willow carr development within a southwest England wetland

Abstract. Woodland colonization on wetlands is considered to have a detrimental effect on their ecological value, even though detailed analysis of this process is lacking. This paper provides an evaluation of the ecological changes resulting from succession of poor fen (base‐poor mire) to willow wet woodland on Goss Moor NNR in Cornwall, UK. Different ages of willow carr were associated with eight understorey communities. During willow colonization, in the ground flora, there was a progressive decrease in poor fen species and an associated increase in woodland species, which appeared to be related to an increase in canopy cover and therefore shade. The most diverse community was found to be the most recent willow and was dominated by poor fen species. The oldest willow was the second most diverse and was associated with a reduction in poor fen species and an increase in woodland species. Architectural features were used successfully to assess the general condition and structure of willow. Tree height and DBH were identified as useful parameters to accurately assess willow age in the field. The implications of active intervention to remove willow in order to conserve the full range of communities within the hydrosere are discussed.