2′-O-Methyl Thiomethyl Modifications in Hammerhead Ribozymes

Abstract

The synthesis of all four phosphoramidites of 2′-O-methylthiomethyl ribonucleosides and their incorporation into hammerhead ribozymes and influence on nuclease stability and catalytic activity is described.     

Improving Heat Stress Tolerance in Camelina sativa and Brassica napus Through Thiourea Seed Priming

Journal of Plant Growth Regulation - Heat stress is a major detriment to crop growth and yield under the current patterns of climate change. Two independent experiments of camelina and canola were...     

Water balance in Rhodnius prolixus during flight: Quantitative aspects of diuresis and its relation to changes in haemolymph and flight-muscle water

The amount of water voided by male Rhodnius prolixus which were flown to exhaustion varied from 0 to over 10% of the initial live weight. It accounted for nearly all of the body water lost during the flight period. Simultaneous measurements on the loss of haemolymph water and an estimate of the amount of faecal water in the excreta indicated that the source of the voided water was primarily the haemolymph. The total water content of the flight muscles changed very little in insects which flew to exhaustion. It is concluded that, despite the diuresis and loss of water, and the considerable reduction in haemolymph volume, dehydration of the flight muscles of male R. prolixus does not occur during these flight periods, and is not a factor contributing to ‘exhaustion’. The possibility that insufficient haemolymph is a factor limiting the duration of flight is discussed.     

A Familiar(ity) Problem: Assessing the Impact of Prerequisites and Content Familiarity on Student Learning

Prerequisites are embedded in most STEM curricula. However, the assumption that the content presented in these courses will improve learning in later courses has not been verified. Because a direct comparison of performance between students with and without required prerequisites is logistically difficult to arrange in a randomized fashion, we developed a novel familiarity scale, and used this to determine whether concepts introduced in a prerequisite course improved student learning in a later course (in two biology disciplines). Exam questions in the latter courses were classified into three categories, based on the degree to which the tested concept had been taught in the prerequisite course. If content familiarity mattered, it would be expected that exam scores on topics covered in the prerequisite would be higher than scores on novel topics. We found this to be partially true for “Very Familiar” questions (concepts covered in depth in the prerequisite). However, scores for concepts only briefly discussed in the prerequisite (“Familiar”) were indistinguishable from performance on topics that were “Not Familiar” (concepts only taught in the later course). These results imply that merely “covering” topics in a prerequisite course does not result in improved future performance, and that some topics may be able to removed from a course thereby freeing up class time. Our results may therefore support the implementation of student-centered teaching methods such as active learning, as the time-intensive nature of active learning has been cited as a barrier to its adoption. In addition, we propose that our familiarity system could be broadly utilized to aid in the assessment of the effectiveness of prerequisites.     

Translation and granule localization of mouse mast cell protease-5. Immunodetection with specific antipeptide Ig.

An antibody to mouse mast cell protease-5 (MMCP-5) was obtained by immunizing a rabbit with a 17-residue synthetic peptide corresponding to the unique amino acid sequence at residues 146 to 162 in this serine protease. After affinity purification, anti-MMCP-5(146-162) Ig reacted in SDS-PAGE immunoblots to recombinant MMCP-5 and to the native MMCP-5 protein present in the lysates of mouse serosal mast cells and the MC5 line of Kirsten sarcoma virus-immortalized mouse mast cells. Immunocytochemical staining localized MMCP-5 to the cytoplasmic granules of serosal mast cells and Kirsten sarcoma virus-immortalized mouse mast cells. Because mouse bone marrow-derived mast cells express abundant amounts of MMCP-5 mRNA, anti-MMCP-5(146-162) Ig was used to study the translation and granule accumulation of this protease when progenitor cells differentiate into these immature mouse mast cells. Maximal expression of MMCP-5 mRNA occurred after bone marrow cells had been cultured for 2 wk in IL-3-rich WEHI-3 cell conditioned medium, and MMCP-5 protein was detected in these cells. However, electron-microscopic analysis with gold-labeled antibody revealed that the amount of MMCP-5 in the individual granules of bone marrow-derived mast cells varied. The highest concentration of MMCP-5 was found in the most electron-dense secretory granules of the cells. These studies demonstrate the ultrastructural localization of the earliest transcribed mouse mast cell chymase, MMCP-5, and its granule accumulation during the differentiation of mouse bone marrow progenitor cells into immature mouse mast cells.     

Tumor necrosis factor-alpha and IFN-gamma expression in human thymus. Localization and overexpression in Down syndrome (trisomy 21).

In vitro studies suggest that TNF-alpha and IFN-gamma regulate thymocyte proliferation, but little evidence exists for the constitutive production of these cytokines in normal human thymus. In paired experiments, we examined frozen sections of postnatal human thymus from four control children and four age-matched children with Down syndrome (DS) (trisomy 21) for TNF-alpha and IFN-gamma mRNA expression using in situ hybridization. We studied thymuses from children with DS because this aneuploid condition is associated with a greatly increased incidence of infection and has abnormal thymic anatomy and patterns of thymocyte maturation. We found cells expressing constitutive levels of TNF-alpha mRNA in the trabeculae, corticomedullary junctions, and medulla of both control and DS thymuses and the number of these cells was an average of 3.9-fold higher in DS thymuses than in age-matched control thymuses. DS thymuses also contained an average of 3 fold higher numbers of cells with mast cell morphology, identified by toluidine blue histologic staining and electron microscopy. In both DS and control thymuses the mast cells colocalized with TNF-alpha mRNA-expressing cells. In addition, TNF-alpha protein- expressing cells, identified by immunohistochemistry, displayed a granular pattern of staining that is characteristic of mast cells. These results suggest that mast cells may be one source of TNF-alpha in human postnatal thymus. Discrete cells expressing IFN-gamma mRNA were distinctly localized to the cortical region of both DS and control thymuses and were 2.4-fold more abundant in DS thymuses than in the controls. Our results demonstrate, for the first time, the constitutive production and location of TNF-alpha and IFN-gamma in postnatal human thymus. The overexpression of both of these cytokines in DS thymuses suggests a dysregulation in cytokine production in DS and may provide an explanation for the abnormal thymic anatomy and thymocyte maturation associated with this syndrome.     

A virus-derived short hairpin RNA confers resistance against sugarcane mosaic virus in transgenic sugarcane

RNA interference (RNAi) is commonly used to produce virus tolerant transgenic plants. The objective of the current study was to generate transgenic sugarcane plants expressing a short hairpin RNAs (shRNA) targeting the coat protein (CP) gene of sugarcane mosaic virus (SCMV). Based on multiple sequence alignment, including genomic sequences of four SCMV strains, a conserved region of ~ 456 bp coat protein (CP) gene was selected as target gene and amplified through polymerase chain reaction (PCR). Subsequently, siRNAs2 and siRNA4 were engineered as stable short hairpin (shRNA) transgenes of 110 bp with stem and loop sequences derived from microRNA (sof-MIR168a; an active regulatory miRNA in sugarcane). These transgenes were cloned in independent RNAi constructs under the control of the polyubiquitin promoter. The RNAi constructs were delivered into two sugarcane cultivars ‘SPF-234 and NSG-311 in independent experiments using particle bombardment. Molecular identification through PCR and Southern blot revealed anti-SCMV positive transgenic lines. Upon mechanical inoculation of transgenic and non-transgenic sugarcane lines with SCMV, the degree of resistance was found variable among the two sugarcane cultivars. For sugarcane cultivar NSG-311, the mRNA expression of the CP–SCMV was reduced to 10% in shRNA2-transgenic lines and 80% in shRNA4-transgenic lines. In sugarcane cultivar SPF-234, the mRNA expression of the CP–SCMV was reduced to 20% in shRNA2-transgenic lines and 90% in shRNA4 transgenic lines, revealing that transgenic plants expressing shRNA4 were almost immune to SCMV infection.