Sonderianin,a furanoid diterpene from Croton sonderianus

From the heartwood of Croton sonderianus was isolated a new furanoid diterpene, sonderianin. The structure was elucidated by spectral analysis, X-ray studies and chemical evidence.     

Molecular characterisation of the caprine (<Emphasis Type="Italic">Capra hircus</Emphasis>) lymphocyte function-associated antigen-1 alpha subunit-encoding cDNA

Background

Lymphocyte function-associated antigen-1 (LFA-1, CD11a/CD18, alpha L beta 2) is required for many cellular adhesive interactions during the immune response.

Results

The Capra hircus CD11a-encoding cDNA was sequenced and compared with its human, murine, rat, bovine and ovine counterparts. Despite some focal differences, it shares all the main characteristics of its known mammalian homologues.

Conclusion

Therefore, along with the caprine CD18-encoding cDNA, which has been available for a few months, the sequence data revealed here will allow the Capra hircus LFA-1 expression in vitro as a tool to explore the specificities of inflammation in the caprine species.

     

The movement protein of cucumber mosaic virus traffics into sieve elements in minor veins of nicotiana clevelandii

The location of the 3a movement protein (MP) of cucumber mosaic virus (CMV) was studied by quantitative immunogold labeling of the wild-type 3a MP in leaves of Nicotiana clevelandii infected by CMV as well as by using a 3a-green fluorescent protein (GFP) fusion expressed from a potato virus X (PVX) vector. Whether expressed from CMV or PVX, the 3a MP targeted plasmodesmata and accumulated in the central cavity of the pore. Within minor veins, the most extensively labeled plasmodesmata were those connecting sieve elements and companion cells. In addition to targeting plasmodesmata, the 3a MP accumulated in the parietal layer of mature sieve elements. Confocal imaging of cells expressing the 3a-GFP fusion protein showed that the 3a MP assembled into elaborate fibrillar formations in the sieve element parietal layer. The ability of 3a-GFP, expressed from PVX rather than CMV, to enter sieve elements demonstrates that neither the CMV RNA nor the CMV coat protein is required for trafficking of the 3a MP into sieve elements. CMV virions were not detected in plasmodesmata from CMV-infected tissue, although large CMV aggregates were often found in the parietal layer of sieve elements and were usually surrounded by 3a MP. These data suggest that CMV traffics into minor vein sieve elements as a ribonucleoprotein complex that contains the viral RNA, coat protein, and 3a MP, with subsequent viral assembly occurring in the sieve element parietal layer.     

Studies on Woloszynskioid Dinoflagellates X: Ultrastructure,Phylogeny and Colour Variation in Tovellia rubescens n. sp. (Dinophyceae)

The external morphology and internal cell fine structure of a new species of Tovelliaceae, Tovellia rubescens n. sp., is described. Phylogenetic analyses based on partial LSU rDNA sequences place the new species in a clade containing Tovellia species that accumulate red pigments and identify T. aveirensis as its closest known relative. Cells of T. rubescens n. sp. were mostly round and had the cingulum located near the middle, with its ends displaced about one cingular width. Small numbers of distinctly flat cells appeared in culture batches; their significance could not be determined. Cells of the new species in culture batches progressively changed from a yellowish‐green, mainly due to chloroplast colour, to a reddish‐brown colour that appeared associated with lipid bodies. The switch to a reddish colour happened earlier in batches grown in medium lacking sources of N or P. Pigment analyses by HPLC‐MS/MS revealed the presence of astaxanthin and astaxanthin‐related metabolites in the new species, but also in T. aveirensis, in which a reddish colour was never observed. The chloroplast arrangement of T. rubescens n. sp. resembled that of T. aveirensis, with lobes radiating from a central pyrenoid complex. The flagellar apparatus and pusular system fell within the general features described from other Tovelliaceae. A row of microtubules interpretable as a microtubular strand of the peduncle was present. Spiny resting cysts with red contents and an ITS sequence identical to that of cultured material of the new species were found in the original locality.     

The activating Ly49W and inhibitory Ly49G NK cell receptors display similar affinities for identical MHC class I ligands

The Ly49 receptor family plays an important role in the regulation of murine natural killer (NK) cell effector function. They recognize cell surface-expressed class I MHC (MHC-I) and are functionally equivalent to the killer Ig-related receptors (KIRs) in human NK cells. Ly49s exist in activating and inhibitory forms with highly homologous extracellular domains, displaying greater variability in the stalk regions. Inhibitory Ly49s can recognize self-MHC-I and therefore mediate tolerance to self. The role of activating Ly49 receptors is less clear. Some activating Ly49 receptors have been shown to recognize MHC-I molecules. The binding affinity of activating Ly49 receptors with MHC-I is currently unknown, and we sought to examine the affinities of two highly related receptors, an activating and an inhibitory Ly49 receptor, for their shared MHC-I ligands. The ectodomain of inhibitory Ly49G of the BALB/c mouse strain is highly similar to the Ly49W activating receptor in the nonobese diabetic (NOD) mouse. Recombinant soluble Ly49G and W were expressed, refolded, and analyzed for binding affinity with MHC-I by surface plasmon resonance. We found that Ly49G and Ly49W bound with similar affinity to the same MHC-I molecules. These results are a first determination of an activating Ly49 receptor affinity for MHC-I and show that, unlike prior results obtained with activating and inhibitory KIR receptors, functional homologues to Ly49 receptors, activating and inhibitory Ly49, can recognize common MHC-I ligands, with similar affinities.     

A feasibility study to identify proteins in the residual Pap test fluid of women with normal cytology by mass spectrometry-based proteomics

Background

The proteomic analysis of body fluids is a growing technology for the identification of protein biomarkers of disease. Given that Papanicolaou tests (Pap tests) are routinely performed on over 30 million women annually in the U.S. to screen for cervical cancer, we examined the residual Pap test fluid as a source of protein for analysis by mass spectrometry (MS). In the liquid-based Pap test, cervical cells are collected from the ectocervix and placed into an alcohol-based fixative prior to staining and pathologic examination. We hypothesized that proteins shed by cells of the female genital tract can be detected in the Pap test fixative by MS-based proteomic techniques. We examined the feasibility of using residual fluid from discarded Pap tests with cytologically “normal” results to optimize sample preparation for MS analysis. The protein composition of the cell-free Pap test fluid was determined by silver staining of sodium dodecyl sulfate -polyacrylamide gels, and the abundance of serum proteins was examined by Western immunoblot using an antibody against human serum albumin. Both pooled and individual samples were trypsin digested and analyzed by two-dimensional MS/MS. Proteins were identified by searching against the Human Uniprot database, and characterized for localization, function and relative abundance.

Results

The average volume of the residual Pap test fluid was 1.5 ml and the average protein concentration was 0.14 mg/ml. By Western immunoblot we showed that the amount of albumin in each sample was significantly reduced compared to normal serum. By MS/MS, we identified 714 unique proteins in pooled Pap test samples and an average of 431 proteins in individual samples. About 40% of the proteins identified were extracellular or localized to the plasma membrane. Almost 20% of the proteins identified were involved in immunity and defense, characteristic of the healthy cervical-vaginal proteome. By merging the protein sets from the individual and pooled Pap test samples, we created a “Normal Pap test Core Proteome” consisting of 153 proteins.

Conclusions

Residual Pap test fluid contains a sufficient amount of protein for analysis by MS and represents a valuable biospecimen source for the identification of protein biomarkers for gynecological diseases.     

Monthly day/night changes and seasonal daily rhythms of sexual steroids in Senegal sole (Solea senegalensis) under natural fluctuating or controlled environmental conditions

In this paper we attempted to investigate the existence of daily fluctuations on plasma sexual steroids (17beta-estradiol, E(2) and testosterone, T) in Senegal sole (Solea senegalensis) females. We described the monthly day/night concentrations and seasonal daily rhythms in animals reared under natural photo- and thermo-period. In addition, the influence of the natural annual fluctuation of the water temperature on the plasma concentration of these steroids was investigated, using one group of Senegal sole under a natural photoperiod, but with an attenuated thermal cycle (around 17-20 degrees C) for one year. Although no significant day/night differences were detected in monthly samplings, the existence of an annual rhythm of E(2) and T (p<0.01) with an acrophase in February was revealed by COSINOR analysis. Maximum values were reached in March for both steroids (6.1+/-1.7 ng mL(-1) at mid-dark, MD and 4.0+/-0.6 ng mL(-1) at mid-light, ML for E2 and 1.4+/-0.4 ng mL(-1) at MD and 0.8+/-0.1 ng mL(-1) at ML for T) in anticipation of the spawning season (May-June). As regards seasonal daily rhythms, the presence of daily oscillations was revealed. At the spring solstice (21st March) a daily rhythm was observed for both steroids (COSINOR, p<0.01), with an acrophase at 20:00 h (E(2)) and at 21:08 h (T). In summer, autumn and winter no daily rhythms were observed due to the low steroid levels at those seasons. When Senegal sole females were submitted to an attenuated annual thermal cycle, the steroid rhythm disappeared (there was no surge in spring, as in the control group) and these fish did not spawn, despite being subjected to natural photoperiod conditions. This result underlined the importance of the natural annual fluctuation of water temperature and photoperiod on the synchronization of the spawning season and on the onset of steroidogenesis.     

Quantitative proteomic analysis by iTRAQ<Superscript>®</Superscript> for the identification of candidate biomarkers in ovarian cancer serum

Background

Ovarian cancer is the most lethal gynecologic malignancy, with the majority of cases diagnosed at an advanced stage when treatments are less successful. Novel serum protein markers are needed to detect ovarian cancer in its earliest stage; when detected early, survival rates are over 90%. The identification of new serum biomarkers is hindered by the presence of a small number of highly abundant proteins that comprise approximately 95% of serum total protein. In this study, we used pooled serum depleted of the most highly abundant proteins to reduce the dynamic range of proteins, and thereby enhance the identification of serum biomarkers using the quantitative proteomic method iTRAQ^®.

Results

Medium and low abundance proteins from 6 serum pools of 10 patients each from women with serous ovarian carcinoma, and 6 non-cancer control pools were labeled with isobaric tags using iTRAQ^® to determine the relative abundance of serum proteins identified by MS. A total of 220 unique proteins were identified and fourteen proteins were elevated in ovarian cancer compared to control serum pools, including several novel candidate ovarian cancer biomarkers: extracellular matrix protein-1, leucine-rich alpha-2 glycoprotein-1, lipopolysaccharide binding protein-1, and proteoglycan-4. Western immunoblotting validated the relative increases in serum protein levels for several of the proteins identified.

Conclusions

This study provides the first analysis of immunodepleted serum in combination with iTRAQ^® to measure relative protein expression in ovarian cancer patients for the pursuit of serum biomarkers. Several candidate biomarkers were identified which warrant further development.

     

Ultrastructure and large subunit rDNA-based phylogeny of Sphaerodinium cracoviense, an unusual freshwater dinoflagellate with a novel type of eyespot

Sphaerodinium cracoviense was collected near Cracow, Poland, and analysed by light microscopy, scanning electron microscopy, and serial-section transmission electron microscopy. Thecae showed a peridinioid type of plate arrangement with unusual numbers in the anterior intercalary and postcingular plate series: 4 and 6, respectively. The apical pore of S. cracoviense differed from the typical arrangement seen in many thecate forms and included a furrow with knob-like protuberances reminiscent of the apical area of some woloszynskioids. The flagellar apparatus included the three microtubular roots that extend to the left of the basal bodies and a striated root connective between the transverse striated root and the longitudinal microtubular root. Both the single-stranded root that associates with the right side of the longitudinal basal body in peridinioids and gonyaulacoids, and the layered connective typical of peridinioids were absent. The eyespot was formed by a layer of vesicle-contained crystal-like units underlain by layers of variably fused globules not bounded by membranes, and represents a novel type. The pusular system included a long canal with a dilated inner portion with radiating tubules. Bayesian and maximum likelihood analyses based on large subunit rDNA placed Sphaerodinium as a sister taxon to a group of woloszynskioids and relatively far from Peridinium and its allies.