Proper targeting and activity of a nonfunctioning thyroid-stimulating hormone receptor (TSHr) combining an inactivating and activating TSHr mutation in one receptor.

Activating mutations of the thyroid-stimulating hormone receptor (TSHr) have been identified as a cause of toxic adenomas. Germline-inactivating TSHr mutations have been described as a cause of congenital hypothyroidism. The effects of combining activating and inactivating mutations within a single receptor was studied. The double mutant T477I/P639S contained an activating TSHr mutation (P639S) together with an inactivating one (T477I). The other one (I486M/P639S) contained two activating mutations. Constructs were expressed in COS-7 cells and basal and TSH-stimulated cyclic AMP (cAMP) accumulation and inositol phosphate (IP) production were determined. The expression at the cell surface was studied both with binding and fluorescence-activated cell scanning analysis. Our results show that the effect of combining the two activating mutations is an increase in the constitutive activity only for the cAMP pathway and not for the IP pathway suggesting that different mutations result in receptor conformations with different relative abilities to couple to Gs-alpha or Gq-alpha. Surprisingly the double mutant containing the T477I behaves as an activating receptor with constitutive activity both for the cAMP and IP pathways. These data show that an inactive form of the TSHr which is trapped inside a cell after transfection is able to gain the membrane surface when combined with an activated form of the receptor.     

Development of a coupled-column liquid chromatographic–tandem mass spectrometric method for the direct determination of betamethasone in urine

Different hyphenated liquid chromatographic (LC) and mass spectrometric (MS) techniques were investigated in order to set-up a method for the fast, direct analysis of betamethasone in hydrolysed and non-hydrolysed urine using large-volume sample injection. After the optimisation of the LC parameters using a traditional UV detector and of the thermospray and mass spectrometric parameters by flow injection, urine samples (0.5 ml) were submitted to analysis by either LC combined with tandem mass spectrometry (MS–MS), coupled-column LC (LC–LC) combined with single quadrupole MS, and LC–LC–MS–MS. Both the three-step configurations (LC–MS–MS and LC–LC–MS) did not provide satisfactory results: loss of sensitivity was noted in the case of LC–MS–MS (likely due to reduced efficiency in the ionisation of betamethasone in the thermospray owing to the presence of large amounts of matrix interference), while in the case of LC–LC–MS a high chemical noise resulting in insufficient selectivity of detection was observed. On the contrary, LC–LC–MS–MS analysis proved to meet the demand of high speed of analysis (sample throughput, 4.5 h⁻¹), selectivity, and sensitivity (LOQ, 1 ng/ml; LOD, 0.2 ng/ml). Notwithstanding the complex analytical system adopted, the developed procedure was manageable and very robust, provided that at the beginning of each analytical session the performance of the system was controlled by checking the retention time of the analytes on the first analytical column with UV detection and by optimising vaporiser temperature of the thermospray by flow injection.     

Oviposition behavior of Anastrepha fraterculus in apple and diel pattern of activities in an apple orchard in Brazil

The oviposition behavior and diel pattern of activities of Anastrepha fraterculus (Wied.) (Diptera: Tephritidae) were observed in an apple orchard and fruit characteristics involved in oviposition preferences were investigated in field cage tests. Fruit size influenced fruit acceptability as an oviposition site by females which did not discriminate among the cultivars Gala, Fuji, and Golden Delicious when same-size fruits were presented simultaneously. Oviposition behavior in apples was basically the same as that described for primary hosts. Hourly census of fly activity indicated that adults did not overnight in the orchard and that they entered the orchard around 1100 h when temperature reached about 21 °C . Bird droppings were an important food item for adults. Behavioral differences between males and females might account for a significant biased sex ratio both in the orchard and at the edge of the native surrounding vegetation. Implications for fruit fly management in Brazilian apple orchards are discussed.     

Isolation of multicellular complexes of follicular dendritic cells and lymphocytes: Immunophenotypical characterization,electron microscopy and culture studies

Summary The morphological and phenotypical features of multicellular complexes formed by follicular dendritic cells and lymphocytes (FDC-LC) isolated from human hyperplastic tonsils and adenoids are described. FDC-LC obtained with this procedure were morphologically and immuno-phenotypically heterogeneous. In one type of FDCLC, probably obtained from germinal centers, the lymphocytes exhibited ultrastructural features of centroblasts and centrocytes. In a second type, likely derived from follicular mantles, the enclosed lymphocytes were small in size and characterized by a condensed chromatin pattern. Similar heterogeneity was observed by immuno-phenotypical analysis, which revealed a prevalence of IgD+, CD3-, MT2+ small lymphocytes in a high proportion of FDC-LC. Both types of FDC-LC contained desmoplakin immunoreactivity in a typical punctate pattern corresponding to intercellular junctions when tested with a specific antibody. These findings confirm the importance of FDC in maintaining the follicular structure and also suggest that the different zones forming lymphoid follicles (mantle zone and germinal center) are formed by lymphocytes gathered in single domains by cytoplasmic processes of FDC. These domains have strong resistance to mechanical stress, such as that used in isolation procedures. FDC-LC have also been maintained as organized multicellular clusters for short periods (more than 48 h) in agarose gel cultures.Supported in part by: Ministero Pubblica Istruzione, Italy, and A.I.R.C., Milano, Italy     

Arctic marine phytobenthos of northern Baffin Island

Global climate change is expected to alter the polar bioregions faster than any other marine environment. This study assesses the biodiversity of seaweeds and associated eukaryotic pathogens of an established study site in northern Baffin Island (72° N), providing a baseline inventory for future work assessing impacts of the currently ongoing changes in the Arctic marine environment. A total of 33 Phaeophyceae, 24 Rhodophyceae, 2 Chlorophyceae, 12 Ulvophyceae, 1 Trebouxiophyceae, and 1 Dinophyceae are reported, based on collections of an expedition to the area in 2009, complemented by unpublished records of Robert T. Wilce and the first‐ever photographic documentation of the phytobenthos of the American Arctic. Molecular barcoding of isolates raised from incubated substratum samples revealed the presence of 20 species of brown seaweeds, including gametophytes of kelp and of a previously unsequenced Desmarestia closely related to D. viridis, two species of Pylaiella, the kelp endophyte Laminariocolax aecidioides and 11 previously unsequenced species of the Ectocarpales, highlighting the necessity to include molecular techniques for fully unraveling cryptic algal diversity. This study also includes the first records of Eurychasma dicksonii, a eukaryotic pathogen affecting seaweeds, from the American Arctic. Overall, this study provides both the most accurate inventory of seaweed diversity of the northern Baffin Island region to date and can be used as an important basis to understand diversity changes with climate change.     

An in vitro study of alkaline phosphatase sensitivity to mixture of aflatoxin B<Subscript>1</Subscript> and fumonisin B<Subscript>1</Subscript> in the hepatopancreas of coastal lagoon wild and farmed shrimp <Emphasis Type="Italic">Litopenaeus vannamei</Emphasis>

This study aimed to establish the combined effect of aflatoxin B₁ (AFB₁) and fumonisin B₁ (FB₁) on wild Litopenaeus vannamei hepatopancreas alkaline phosphatase (AP) activity compared with that of farmed shrimp. AP activity in hepatopancreas extract was confirmed by several specific inhibitor assays. AP activity of wild shrimp was higher than that of farmed shrimp (p?<?0.05). However, AP activity from both wild and farmed shrimp was inhibited when incubated with AFB₁ and FB₁. The greatest inhibition occurred when AP was incubated with a mixture of AFB₁ and FB₁. The IC₅₀ for AFB₁ on AP activity of wild and farmed shrimp hepatopancreases was 0.790 and 0.398 μg/mL, respectively. The IC₅₀ of FB₁ was 0.87 μg/mL for wild shrimp and 0.69 μg/mL for farmed shrimp. These results suggest that, at the mycotoxins concentrations used in the study, AP from farmed L. vannamei was sensitive to the presence of both mycotoxins; however, AP is more sensitive to the combination of AFB₁?+?FB₁ suggesting a possible synergistic or potentiating inhibitory effect.     

A checklist of helminth parasites of Elasmobranchii in Mexico

A comprehensive and updated summary of the literature and unpublished records contained in scientific collections on the helminth parasites of the elasmobranchs from Mexico is herein presented for the first time. At present, the helminth fauna associated with Elasmobranchii recorded in Mexico is composed of 132 (110 named species and 22 not assigned to species), which belong to 70 genera included in 27 families (plus 4 incertae sedis families of cestodes). These data represent 7.2% of the worldwide species richness. Platyhelminthes is the most widely represented, with 128 taxa: 94 of cestodes, 22 of monogeneans and 12 of trematodes; Nematoda and Annelida: Hirudinea are represented by only 2 taxa each. These records come from 54 localities, pertaining to 15 states; Baja California Sur (17 sampled localities) and Baja California (10), are the states with the highest species richness: 72 and 54 species, respectively. Up to now, 48 elasmobranch species have been recorded as hosts of helminths in Mexico; so, approximately 82% of sharks and 67% of rays distributed in Mexican waters lack helminthological studies. The present list provides the host, distribution (with geographical coordinates), site of infection, accession number in scientific collections, and references for the parasites. A host-parasite list is also provided.