Morphological and cytochemical studies of circulating Hodgkin's and Reed-Sternberg cells

Morphological and cytochemical studies of circulating neoplastic cells were carried out in a patient who presented a preterminal leukaemic phase of Hodgkin's disease (HD). Three types of abnormal cells were found in the peripheral blood: abnormal mononuclear cells, Hodgkin's cells and Reed-Sternberg cells. All neoplastic cells were cytochemically negative to Sudan black B, peroxidase and alkaline phosphatase. Some neoplastic cells were positive to PAS and all were positive to acid phosphatase, alpha-naphthylacetate esterase and beta-glucuronidase. The origin of the neoplastic population in HD is discussed.     

Fatty Acid‐Derived Pro‐Toxicants of the Rat Selective Toxicant Norbormide

Norbormide [5‐(α‐hydroxy‐α‐2‐pyridylbenzyl)‐7‐(α‐2‐pyridylbenzylidene)‐5‐norbornene‐2,3‐dicarboximide] (NRB), an existing but infrequently used rodenticide, is known to be uniquely toxic to rats but relatively harmless to other rodents and mammals. However, as an acute vasoactive, NRB has a rapid onset of action which makes it relatively unpalatable to rats, often leading to sublethal uptake and accompanying bait shyness. A series of NRB‐derived pro‐toxicants ( 3a  –  i , 4a  –  i , and 5a  –  i ) were prepared in an effort to ‘mask’ this acute response and improve both palatability and efficacy. Their synthesis, in vitro biological evaluation (vasocontractile response in rat vasculature, stability in selected rat media) and palatability/efficacy in Sprague–Dawley, wild Norway, and wild ship rats is described. Most notably, pro‐toxicant 3d was revealed to be free of all pre‐cleavage vasoconstrictory activity in rat caudal artery and was subsequently demonstrated to release NRB in the presence of rat blood, liver, and pancreatic enzymes. Moreover, it consistently displayed a high level of acceptance by rats in a two‐choice bait‐palatability and efficacy trial, with accompanying high mortality. On this evidence, fatty acid ester prodrugs would appear to offer a promising platform for the further development of NRB‐derived toxicants with enhanced palatability and efficacy profiles.     

Nutrient availability and nutrient use efficiency in plants growing in the transition zone between land and water

The transition zone between terrestrial and freshwater habitats is highly dynamic, with large variability in environmental characteristics. Here, we investigate how these characteristics influence the nutritional status and performance of plant life forms inhabiting this zone. Specifically, we hypothesised that: (i) tissue nutrient content differs among submerged, amphibious and terrestrial species, with higher content in submerged species; and (ii) PNUE gradually increases from submerged over amphibious to terrestrial species, reflecting differences in the availability of N and P relative to inorganic C across the land–water ecotone. We found that tissue nutrient content was generally higher in submerged species and C:N and C:P ratios indicated that content was limiting for growth for ca. 20% of plant individuals, particularly those belonging to amphibious and terrestrial species groups. As predicted, the PNUE increased from submerged over amphibious to terrestrial species. We suggest that this pattern reflects that amphibious and terrestrial species allocate proportionally more nutrients into processes of importance for photosynthesis at saturating CO₂ availability, i.e. enzymes involved in substrate regeneration, compared to submerged species that are acclimated to lower availability of CO₂ in the aquatic environment. Our results indicate that enhanced nutrient loading may affect relative abundance of the three species groups in the land–water ecotone of stream ecosystems. Thus, species of amphibious and terrestrial species groups are likely to benefit more from enhanced nutrient availability in terms of faster growth compared to aquatic species, and that this can be detrimental to aquatic species growing in the land–water ecotone, e.g. Ranunculus and Callitriche.     

Immunohistochemical localization of irisin in skin,eye, and thyroid and pineal glands of the crested porcupine (Hystrix cristata)

Irisin was first identified in muscle cells. We detected irisin immunoreactivity in various organs of the crested porcupine (Hystrix cristata). In the epidermis, irisin immunoreactivity was localized mainly in stratum basale, stratum spinosum and stratum granulosum layers; immunoreactivity was not observed in the stratum corneum. In the dermis, irisin was found in the external and internal root sheath, cortex and medulla of hair follicles, and in sebaceous glands. Irisin immunoreactivity was found in the neural retina and skeletal muscle fibers associated with the eye. The pineal and thyroid glands also exhibited irisin immunoreactivity.     

Covalent attachment of proteins to solid supports and surfaces via Sortase-mediated ligation

Background

There is growing interest in the attachment of proteins to solid supports for the development of supported catalysts, affinity matrices, and micro devices as well as for the development of planar and bead based protein arrays for multiplexed assays of protein concentration, interactions, and activity. A critical requirement for these applications is the generation of a stable linkage between the solid support and the immobilized, but still functional, protein.

Methodology

Solid supports including crosslinked polymer beads, beaded agarose, and planar glass surfaces, were modified to present an oligoglycine motif to solution. A range of proteins were ligated to the various surfaces using the Sortase A enzyme of S. aureus. Reactions were carried out in aqueous buffer conditions at room temperature for times between one and twelve hours.

Conclusions

The Sortase A transpeptidase of S. aureus provides a general, robust, and gentle approach to the selective covalent immobilization of proteins on three very different solid supports. The proteins remain functional and accessible to solution. Sortase mediated ligation is therefore a straightforward methodology for the preparation of solid supported enzymes and bead based assays, as well as the modification of planar surfaces for microanalytical devices and protein arrays.     

Identification by denaturing high-performance liquid chromatography of numerous polymorphisms in a candidate region for multiple sclerosis susceptibility

Genetic association analysis of candidate regions where evidence of linkage has accumulated is becoming a key issue in the study of complex diseases. A high density of markers, at least one per centimorgan, is required to improve the chances of observing linkage disequilibrium with disease alleles. A recently available single nucleotide polymorphism (SNP) map designed to cover the whole genome provides an average density of one marker per 2 cM. In the present study we show that the number of markers can be approximately doubled in a selected region, thus reaching a density suitable for association studies, by applying a completely automated technique for polymorphism detection, denaturing high-performance liquid chromatography (DHPLC). A systematic search for SNPs was performed in the region 5ptel-q13, where weak but convergent evidence for linkage with multiple sclerosis has accumulated. Screening for polymorphisms was performed on 124 sequence tagged sites (STSs) in the 3'UTR ends of expressed sequence tags totaling about 30,000 bp. Thirty SNPs in 28 STSs were found with less than 10% overlap with the markers already detected in the same region. The data confirm the validity of the approach using DHPLC on expressed gene sequences tagged by a set of standard commercially available primers.     

Expression and functional characterization of SCaMPER: a sphingolipid-modulated calcium channel of cardiomyocytes

Calciumchannels are important in a variety of cellular events including musclecontraction, signaling, proliferation, and apoptosis.Sphingolipids have been recognized as mediators of intracellularcalcium release through their actions on a calcium channel,sphingolipid calcium release-mediating protein of the endoplasmicreticulum (SCaMPER). The current study investigates the expression andfunction of SCaMPER in cardiomyocytes. Northern analyses and RT-PCRcloning and sequencing revealed SCaMPER expression in both human andrat cardiac tissue. Immunofluorescence and Western blot analysesdemonstrated that SCaMPER is abundant in cardiac tissue and islocalized to the sarcotubular junction. This was confirmed by thecolocalization of SCaMPER with dihydropyridine and ryanodine receptorsby confocal microscopy. Purified T tubules were shown to containSCaMPER and immunoelectron micrographs suggested that SCaMPER islocated to the junctional T tubules, but a junctional SR localizationcannot be ruled out. The sphingolipid ligand for SCaMPER,sphingosylphosphorylcholine (SPC), initiated calcium release from thecardiomyocyte SR. Importantly, antisense knockdown of SCaMPER mRNAproduced a substantial reduction of sphingolipid-induced calciumrelease, suggesting that SCaMPER is a potentially important calciumchannel of cardiomyocytes.

     

The stress-induced MAP kinase p38 regulates endocytic trafficking via the GDI:Rab5 complex

Early endocytic membrane traffic is regulated by the small GTPase Rab5, which cycles between GTP- and GDP-bound states as well as between membrane and cytosol. The latter cycle depends on GDI, which functions as a Rab vehicle in the aqueous environment of the cytosol. Here, we report that formation of the GDI:Rab5 complex is stimulated by a cytosolic factor that we purified and then identified as p38 MAPK. We find that p38 regulates GDI in the cytosolic cycle of Rab5 and modulates endocytosis in vivo. Our observations reveal the existence of a cross-talk between endocytosis and the p38-dependent stress response, thus providing molecular evidence that endocytosis can be regulated by the environment.     

Comparison of two cysteine endopeptidases from latices of Morrenia brachystephana Griseb. and Morrenia odorata (Hook et Arn.) Lindley (Asclepiadaceae)

The properties of morrenain b II, a proteinase isolated from the latex of Morrenia brachystephana, were compared with those of morrenain o II, a proteinase obtained from the latex of Morrenia odorata. Both peptidases were purified to homogeneity by acetone precipitation followed by cation exchange chromatography. The enzymes have pI values higher than 9.3 and similar molecular masses (close to 26 kDa) as determined by SDS-PAGE. They display maximum proteolytic activity within an alkaline pH range, and also exhibit esterolytic activity. The N-terminal sequences of morrenain o II and morrenain b II show a high degree of homology between each other and to other cysteine plant proteinases.