Studies on the surface coat of Paramecium aurelia

Summary The plasma membrane of Paramecium aurelia is covered with a ruthenium red stainable surface coat. Results obtained after digestion with pronase, trypsin and neuraminidase suggest the glycoprotein nature of this structure. Lipid extraction also affects the surface coat forming material. The results are consistent with the model proposed by Ginsburg and Kobata dealing with spatial configuration of the surface coat components.Authors are grateful to Mrs. D. Kucharczyk for very efficient technical assistence, to Mrs. Z. Kaminska for sectioning the material and Mr. A. Renski for help with the electron microscope service.     

Immunocytochemical study of the glucocorticoid receptor in rat liver nuclei after hyperthermic stress

The presence of glucocorticoid receptors (GR) in rat liver nuclei over a 24 h time period following hyperthermic stress at 41 degrees C was immunocytologically studied using unfixed nuclear smears. Liver nuclei in unstressed animals were found to be immunonegative for GR. However, intense GR immunopositivity followed by a subsequent gradual decrease in receptor levels was observed in the nuclei of test animals during the first 2 h after stress. This stress-related increase in the receptor nuclear level was greater than the increase seen after dexamethasone administration. These results suggest that hyperthermic stress could potentiate the hormonal stimulation of receptor nuclear translocation.     

‘Click’ chemistry synthesis and capillary electrophoresis study of 1,4‐linked 1,2,3‐triazole AZT‐systemin conjugate

The Cu(I) catalyzed Huisgen 1,3‐dipolar azide‐alkyne cycloaddition (CuAAC) was applied for a nucleoside‐peptide bioconjugation. Systemin (Sys), an 18‐aa plant signaling peptide naturally produced in response to wounding or pathogen attack, was chemically synthesized as its N‐propynoic acid functionalized analog (Prp‐Sys) using the SPPS. Next, CuAAC was applied to conjugate Prp‐Sys with 3′‐azido‐2′,3′‐dideoxythymidine (AZT), a model cargo molecule. 1,4‐Linked 1,2,3‐triazole AZT‐Sys conjugate was designed to characterize the spreading properties and ability to translocate of cargo molecules of systemin. CuAAC allowed the synthesis of the conjugate in a chemoselective and regioselective manner, with high purity and yield. The presence of Cu(I) ions generated in situ drove the CuAAC reaction to completion within a few minutes without any by‐products. Under typical separation conditions of phosphate ‘buffer’ at low pH and uncoated fused bare‐silica capillary, an increasing peak intensity assigned to triazole‐linked AZT‐Sys conjugate was observed using capillary electrophoresis (CE) during CuAAC. CE analysis showed that systemin peptides are stable in tomato leaf extract for up to a few hours. CE‐ESI‐MS revealed that the native Sys and its conjugate with AZT are translocated through the tomato stem and can be directly detected in stem exudates. The results show potential application of systemin as a transporter of low molecular weight cargo molecules in tomato plant and of CE method to characterize a behavior of plant peptides and its analogs. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Karyotype and nuclear DNA content of hexa-, octo-, and duodecaploid lines of Bromus subgen. Ceratochloa

The subgenus Ceratochloa of the genus Bromus includes a number of closely related allopolyploid forms or species that present a difficult taxonomic problem. The present work combines data concerning chromosome length, heterochromatin distribution and nuclear genome size of different 6x, 8x and 12x accessions in this subgenus. Special attention is paid to the karyotype structure and genomic constitution of duodecaploid plants recently found in South America. Hexaploid lineages possess six almost indistinguishable genomes and a nuclear DNA content between 12.72 pg and 15.10 pg (mean 1Cx value = 2.32 pg), whereas octoploid lineages contain the same six genomes (AABBCC) plus two that are characterized by longer chromosomes and a greater DNA content (1Cx = 4.47 pg). Two duodecaploid accessions found in South America resemble each other and apparently differ from the North American duodecaploid B. arizonicus as regards chromosome size and nuclear DNA content (40.00 and 40.50 pg vs. 27.59 pg). These observations suggest that the South American duodecaploids represent a separate evolutionary lineage of the B. subgenus Ceratochloa, unrecognized heretofore.     

Optimization of 131I ablation in patients with differentiated thyroid carcinoma: comparison of early outcomes of treatment with 100 mCi versus 60 mCi

INTRODUCTION: The aim of this study was to compare the early outcomes between two groups of patients with differentiated thyroid carcinoma (DTC) who received 60 or 100 mCi of (131)I for remnant ablation. MATERIAL AND METHODS: 224 DTC patients with primary tumor > 1 cm of diameter or multifocal were randomised into prospective clinical trial. Patients with extrathyroideal extension of primary tumor and nodal metastases or M1 were not enrolled. 99 patients received 60 mCi, and 125--100 mCi of radioiodine as the first ablative dose. RESULTS: The effectiveness of thyroid ablation was evaluated after one year, during endogenous TSH (thyroid stimulating hormone) stimulation, and after two years during Lthyroxine therapy. Whole body scintigraphy (WBS) was performed under thyroxine withdrawal and thyroglobulin serum level was assessed. Distant micrometastases were detected in 9.8% of patients by post-therapy WBS, 11 patients in group A treated with 60 mCi and 11 in group B treated with 100 mCi. In other patients no symptoms of persistent disease were detected. At one year follow up full remission was diagnosed in 176 patients: 76 in group A and 100 in group B. The remaining ones, 13.3% and 11.2% respectively, received the second course of (131)I for remnant ablation. There were no statistically significant differences in Tg (thyroglobulin) serum level either 12 or 24 months after 131I treatment. CONCLUSIONS: Our evaluation of early efficacy of adjuvant radioiodine treatment in low risk DTC patients shows no differences between two radioiodine activities - 60 and 100 mCi in relation to thyroid ablation. Thus, the activity of 60 mCi is recommended.     

Caveola-dependent endocytic entry of amphotropic murine leukemia virus

Early results suggested that the amphotropic murine leukemia virus (A-MLV) does not enter cells via endocytosis through clathrin-coated pits and this gammaretrovirus has therefore been anticipated to fuse directly with the plasma membrane. However, here we present data implicating a caveola-mediated endocytic entry route for A-MLV via its receptor Pit2. Caveolae belong to the cholesterol-rich microdomains characterized by resistance to nonionic detergents such as Triton X-100. Extraction of murine fibroblastic NIH 3T3 cells in cold Triton X-100 showed the presence of the A-MLV receptor Pit2 in detergent-insoluble microdomains. Using coimmunoprecipitation of cell extracts, we were able to demonstrate direct association of Pit2 with caveolin-1, the structural protein of caveolae. Other investigations revealed that A-MLV infection in contrast to vesicular stomatitis virus infection is a slow process (t(1/2) approximately 5 h), which is dependent on plasma membrane cholesterol but independent of NH4Cl treatment of cells; NH4Cl impairs entry via clathrin-coated pits. Furthermore, expression of dominant-negative caveolin-1 decreased the susceptibility to infection via Pit2 by approximately 70%. These results show that A-MLV can enter cells via a caveola-dependent entry route. Moreover, increase in A-MLV infection by treatment with okadaic acid as well as entry of fusion-defective fluorescent A-MLV virions in NIH 3T3 cells further confirmed our findings and show that A-MLV can enter mouse fibroblasts via an endocytic entry route involving caveolae. Finally, we also found colocalization of fusion-defective fluorescent A-MLV virions with caveolin-1 in NIH 3T3 cells. This is the first time substantial evidence has been presented implicating the existence of a caveola-dependent endocytic entry pathway for a retrovirus.     

3′-End processing of histone pre-mRNAs in Drosophila: U7 snRNP is associated with FLASH and polyadenylation factors

3′-End cleavage of animal replication-dependent histone pre-mRNAs is controlled by the U7 snRNP. Lsm11, the largest component of the U7-specific Sm ring, interacts with FLASH, and in mammalian nuclear extracts these two proteins form a platform that recruits the CPSF73 endonuclease and other polyadenylation factors to the U7 snRNP. FLASH is limiting, and the majority of the U7 snRNP in mammalian extracts exists as a core particle consisting of the U7 snRNA and the Sm ring. Here, we purified the U7 snRNP from Drosophila nuclear extracts and characterized its composition by mass spectrometry. In contrast to the mammalian U7 snRNP, a significant fraction of the Drosophila U7 snRNP contains endogenous FLASH and at least six subunits of the polyadenylation machinery: symplekin, CPSF73, CPSF100, CPSF160, WDR33, and CstF64. The same composite U7 snRNP is recruited to histone pre-mRNA for 3′-end processing. We identified a motif in Drosophila FLASH that is essential for the recruitment of the polyadenylation complex to the U7 snRNP and analyzed the role of other factors, including SLBP and Ars2, in 3′-end processing of Drosophila histone pre-mRNAs. SLBP that binds the upstream stem–loop structure likely recruits a yet-unidentified essential component(s) to the processing machinery. In contrast, Ars2, a protein previously shown to interact with FLASH in mammalian cells, is dispensable for processing in Drosophila. Our studies also demonstrate that Drosophila symplekin and three factors involved in cleavage and polyadenylation—CPSF, CstF, and CF I_m—are present in Drosophila nuclear extracts in a stable supercomplex.