Identification of tissue-specific microRNAs from mouse

MicroRNAs (miRNAs) are a new class of noncoding RNAs, which are encoded as short inverted repeats in the genomes of invertebrates and vertebrates. It is believed that miRNAs are modulators of target mRNA translation and stability, although most target mRNAs remain to be identified. Here we describe the identification of 34 novel miRNAs by tissue-specific cloning of approximately 21-nucleotide RNAs from mouse. Almost all identified miRNAs are conserved in the human genome and are also frequently found in nonmammalian vertebrate genomes, such as pufferfish. In heart, liver, or brain, it is found that a single, tissue-specifically expressed miRNA dominates the population of expressed miRNAs and suggests a role for these miRNAs in tissue specification or cell lineage decisions. Finally, a miRNA was identified that appears to be the fruitfly and mammalian ortholog of C. elegans lin-4 stRNA.     

Effect of H. pylori on the expression of TRAIL, FasL and their receptor subtypes in human gastric epithelial cells and their role in apoptosis

BACKGROUND AND AIMS: In the human stomach expression of TNF-related apoptosis inducing ligand (TRAIL) and its receptors and the modulatory role of Helicobacter pylori are not well described. Therefore, we investigated the effect of H. pylori on the expression of TRAIL, FasL and their receptors (TRAIL-R1-R4, Fas) in gastric epithelial cells and examined their role in apoptosis. MATERIALS AND METHODS: mRNA and protein expression of TRAIL, FasL and their receptors were analyzed in human gastric epithelial cells using RT-PCR, Western blot, and immunohistochemistry. Gastric epithelial cells were incubated with FasL, TRAIL and/or H. pylori, and effects on expression, cell viability and epithelial apoptosis were monitored. Apoptosis was analyzed by histone ELISA, DAPI staining and immunohistochemistry. RESULTS: TRAIL, FasL and their receptor subtypes were expressed in human gastric mucosa, gastric epithelial cell primary cultures and gastric cancer cells. TRAIL, FasL and H. pylori caused a time- and concentration-dependent induction of DNA fragmentation in gastric cancer cells with synergistic effects. In addition, H. pylori caused a selective up-regulation of TRAIL, TRAIL-R1 and Fas mRNA and protein expression in gastric cancer cells. CONCLUSIONS: Next to FasL and Fas, TRAIL and all of its receptor subtypes are expressed in the human stomach and differentially modulated by H. pylori. TRAIL, FasL and H. pylori show complex interaction mediating apoptosis in human gastric epithelial cells. These findings might be important for the understanding of gastric epithelial cell kinetics in patients with H. pylori infection.     

Combined Ki-67 and feulgen stain for morphometric determination of the Ki-67 labelling index

In this note we present a combined Ki-67 and Feulgen stain for morphometric determination of the Ki-67 labelling index. The immunohistochemical part of this double staining technique is based on the alkaline-phosphatase-anti-alkaline-phosphatase (APAAP) method, visualizing the enzyme activity by the nitro-blue-tetrazolium chloride (NBT)/bromo-chloro-3-indolyl-phosphate (BCIP) technique. The NBT/BCIP complex resists the hydrolytic activity of the Feulgen stain. The staining method presented allows semi-automatic determination of both the total nucleus-area as well as the Ki-67 positive nucleus-area using a morphometric computer system. The Ki-67 labelling index thus achieved is based on the relative nuclear area of Ki-67 positive nuclei and is clearly more precise and efficient than the counting method using an ocular grid.     

Structural basis for membrane fusion by enveloped viruses.

Enveloped viruses such as HIV-1, influenza virus, and Ebola virus express a surface glycoprotein that mediates both cell attachment and fusion of viral and cellular membranes. The membrane fusion process leads to the release of viral proteins and the RNA genome into the host cell, initiating an infectious cycle. This review focuses on the HIV-1 gp41 membrane fusion protein and discusses the structural similarities of viral membrane fusion proteins from diverse families such as Retroviridae (HIV-1), Orthomyxoviridae (influenza virus), and Filoviridae (Ebola virus). Their structural organization suggests that they have all evolved to use a similar strategy to promote fusion of viral and cellular membranes. This observation led to the proposal of a general model for viral membrane fusion, which will be discussed in detail.     

Bacteriorhodopsin,boundary lipid and protein conformers. A spin label study

A spin label study, as a function of temperature, has been made with the bacteriorhodopsin membrane using a stearic acid spin label. The ESR spectra show a strong variation with temperature and the presence of isosbestic points. The spectra are interpreted as indicating the presence of a two-component system with an activation energy (approx. 14 kcal/mol) corresponding to a protein conformational change. This activation energy is similar to that deduced from recent flash photolysis studies.It is concluded that the spin label is sensitive to the temperature-dependent protein conformational change in this membrane system.     

Long term effects of manure, charcoal and mineral fertilization on crop production and fertility on a highly weathered Central Amazonian upland soil

Application of organic fertilizers and charcoal increase nutrient stocks in the rooting zone of crops, reduce nutrient leaching and thus improve crop production on acid and highly weathered tropical soils. In a field trial near Manaus (Brazil) 15 different amendment combinations based on equal amounts of carbon (C) applied through chicken manure (CM), compost, charcoal, and forest litter were tested during four cropping cycles with rice (Oryza sativa L.) and sorghum (Sorghum bicolor L.) in five replicates. CM amendments resulted in the highest (P < 0.05) cumulative crop yield (12.4 Mg ha⁻¹) over four seasons. Most importantly, surface soil pH, phosphorus (P), calcium (Ca), and magnesium (Mg) were significantly enhanced by CM. A single compost application produced fourfold more grain yield (P < 0.05) than plots mineral fertilized in split applications. Charcoal significantly improved plant growth and doubled grain production if fertilized with NPK in comparison to the NPK-fertilizer without charcoal (P < 0.05). The higher yields caused a significantly greater nutrient export in charcoal-amended fields, but available nutrients did not decrease to the same extent as on just mineral fertilized plots. Exchangeable soil aluminum (Al) was further reduced if mineral fertilizer was applied with charcoal (from 4.7 to 0 mg kg⁻¹). The resilience of soil organic matter (SOM) in charcoal amended plots (8 and 4% soil C loss, mineral fertilized or not fertilized, respectively) indicates the refractory nature of charcoal in comparison to SOM losses over 20 months in CM (27%), compost amended (27%), and control plots (25% loss).     

Isolation and characterization of rtcs, a maize mutant deficient in the formation of nodal roots

The root system of maize consists of the primary root and a variable number of lateral seminal-, crown- and brace roots. Except for the primary root and some minor roots forming at the mesocotyl, all other roots grow out of nodal regions, namely, the embryogenic scutellar node and the underground—as well as the lower above-ground stem nodes. Besides their role in water and nutrient uptake, some of these roots (crown- and brace roots) are essential for the lodging resistance of the plants. This property of the crown roots has now been successfully used for screening a segregating F₂ population of a cross between a flint inbred line and an En transposon line. Two allelic root-deficient mutants have been isolated and have been designated rtcs-1 and rtcs-2 for their complete lack of formation of c rown- and lateral s eminal roots. They survive by the ability of the primary root to support the growth of the developing plant. The monogenic and recessive mutants appear to be affected in an early root-forming function since no primordia are formed either in the case of embryo-borne lateral seminal or stem-derived crown roots. The Rtcs locus could be mapped to the short arm of chromosome 1 with the help of a co-segregating RAPD marker. The effect of the mutation seems to be highly specific since no pleiotropic effects on other parts of the plants have been observed. The formation of adventitious roots can, however, still be induced in the mesocotyl region of the mutant.     

The EndoPredict Gene-Expression Assay in Clinical Practice - Performance and Impact on Clinical Decisions

The validated EndoPredict assay is a novel tool to predict the risk of metastases of patients with estrogen receptor positive, HER2 negative breast cancer treated with endocrine therapy alone. It has been designed to integrate genomic and clinical information and includes clinico-pathological factors such as tumor size and nodal status. The test is feasible in a decentral setting in molecular pathology laboratories. In this project, we investigated the performance of this test in clinical practice, and performed a retrospective evaluation of its impact on treatment decisions in breast cancer. During one year, EndoPredict assays from 167 patients could be successfully performed. For retrospective evaluation of treatment decisions, a questionnaire was sent to the clinical partner. Regarding the molecular EP class, samples from 56 patients (33.5%) had a low-risk, whereas 111 patients (66.5%) showed a high-risk gene profile. After integration of the clinicopathological factors the combined clinical and molecular score (EPclin) resulted in a low-risk group of 77 patients (46.4%), while 89 (53.6%) had a high risk EPclin score. The EPclin-based estimated median 10-year-risk for metastases with endocrine therapy alone was 11% for the whole cohort. The median handling time averaged three days (range: 0 to 11 days), 59.3% of the tests could be performed in three or less than three days. Comparison of pre- and post-test therapy decisions showed a change of therapy in 37.7% of patients. 16 patients (12.3%) had a change to an additional chemotherapy while 25.4% of patients (n = 33) changed to an endocrine therapy alone. In 73 patients (56.2%) no change of therapy resulted. In 6.1% of patients (n = 8), the patients did not agree to the recommendation of the tumor board. Our results show that the EndoPredict assay could be routinely performed in decentral molecular pathology laboratories and the results markedly change treatment decisions.