Secondary heterologous dengue infection risk: Disequilibrium between immune regulation and inflammation?

Increased serum levels of cytokines released by cells of the immune response have been detected in patients suffering from dengue disease. Likewise, secondary infections by a different dengue virus serotype result in a highest risk of development of the severe dengue disease. Both findings suggest that the memory immune response is one of the key players in the pathogenesis of this disease. Here we take advantage of the particular Cuban epidemiological situation in dengue to analyze a broad spectrum of cell-mediated immune response mediators at mRNA and protein level. Evidences for a regulatory immune pattern in homologous (TGF-β, IL-10) vs. pro-inflammatory pattern (IFN-γ, TNF-α) in heterologous dengue virus re-challenge were found, suggesting a possible association with the higher incidence of severe dengue cases in the latter case.     

Exploration of the Arrest Peptide Sequence Space Reveals Arrest-enhanced Variants

Translational arrest peptides (APs) are short stretches of polypeptides that induce translational stalling when synthesized on a ribosome. Mechanical pulling forces acting on the nascent chain can weaken or even abolish stalling. APs can therefore be used as in vivo force sensors, making it possible to measure the forces that act on a nascent chain during translation with single-residue resolution. It is also possible to score the relative strengths of APs by subjecting them to a given pulling force and ranking them according to stalling efficiency. Using the latter approach, we now report an extensive mutagenesis scan of a strong mutant variant of the Mannheimia succiniciproducens SecM AP and identify mutations that further increase the stalling efficiency. Combining three such mutations, we designed an AP that withstands the strongest pulling force we are able to generate at present. We further show that diproline stretches in a nascent protein act as very strong APs when translation is carried out in the absence of elongation factor P. Our findings highlight critical residues in APs, show that certain amino acid sequences induce very strong translational arrest and provide a toolbox of APs of varying strengths that can be used for in vivo force measurements.     

Fluorescence imaging with two-photon evanescent wave excitation

We demonstrate broad-field, non-scanning, two-photon excitation fluorescence (2PEF) close to a glass/cell interface by total internal reflection of a femtosecond-pulsed infrared laser beam. We exploit the quadratic intensity dependence of 2PEF to provide non-linear evanescent wave (EW) excitation in a well-defined sample volume and to eliminate scattered background excitation. A simple model is shown to describe the resulting 2PEF intensity and to predict the effective excitation volume in terms of easily measurable beam, objective and interface properties. We demonstrate non-linear evanescent wave excitation at 860 nm of acridine orange-labelled secretory granules in live chromaffin cells, and excitation at 900 nm of TRITC-phalloidin-actin/GPI-GFP double-labelled fibroblasts. The confined excitation volume and the possibility of simultaneous multi-colour excitation of several fluorophores make EW 2PEF particularly advantageous for quantitative microscopy, imaging biochemistry inside live cells, or biosensing and screening applications in miniature high-density multi-well plates.Abbreviations 1PEF one-photon excited fluorescence - 2PEF two-photon excited fluorescence - APD avalanche photo diode - CHO Chinese hamster ovary - DMEM Dulbecco's modified Eagle's medium - EGFP enhanced green fluorescent protein - EW evanescent wave - FCS fetal calf serum - GPI glycosylphosphatidylinositol - TIR total internal reflectionThis paper is dedicated to the memory of Prof. Horst Harreis (1940–2002)     

The Medicago truncatula CRE1 cytokinin receptor regulates lateral root development and early symbiotic interaction with Sinorhizobium meliloti

Legumes develop different types of lateral organs from their primary root, lateral roots and nodules, the latter depending on a symbiotic interaction with Sinorhizobium meliloti. Phytohormones have been shown to function in the control of these organogeneses. However, related signaling pathways have not been identified in legumes. We cloned and characterized the expression of Medicago truncatula genes encoding members of cytokinin signaling pathways. RNA interference of the cytokinin receptor homolog Cytokinin Response1 (Mt CRE1) led to cytokinin-insensitive roots, which showed an increased number of lateral roots and a strong reduction in nodulation. Both the progression of S. meliloti infection and nodule primordia formation were affected. We also identified two cytokinin signaling response regulator genes, Mt RR1 and Mt RR4, which are induced early during the symbiotic interaction. Induction of these genes by S. meliloti infection is altered in mutants affected in the Nod factor signaling pathway; conversely, cytokinin regulation of the early nodulin Nodule Inception1 (Mt NIN) depends on Mt CRE1. Hence, cytokinin signaling mediated by a single receptor, Mt CRE1, leads to an opposite control of symbiotic nodule and lateral root organogenesis. Mt NIN, Mt RR1, and Mt RR4 define a common pathway activated during early S. meliloti interaction, allowing crosstalk between plant cytokinins and bacterial Nod factors signals.     

Molecular basis of the head‐to‐tail assembly of giant muscle proteins obscurin‐like 1 and titin

Large filament proteins in muscle sarcomeres comprise many immunoglobulin‐like domains that provide a molecular platform for self‐assembly and interactions with heterologous protein partners. We have unravelled the molecular basis for the head‐to‐tail interaction of the carboxyl terminus of titin and the amino‐terminus of obscurin‐like‐1 by X‐ray crystallography. The binary complex is formed by a parallel intermolecular β‐sheet that presents a novel immunoglobulin‐like domain‐mediated assembly mechanism in muscle filament proteins. Complementary binding data show that the assembly is entropy‐driven rather than dominated data by specific polar interactions. The assembly observed leads to a V‐shaped zipper‐like arrangement of the two filament proteins.     

GENETIC DATA HINT AT A COMMON DONOR REGION FOR INVASIVE ATLANTIC AND PACIFIC POPULATIONS OF GRACILARIA VERMICULOPHYLLA (GRACILARIALES,RHODOPHYTA)1

Gracilaria vermiculophylla (Ohmi) Papenf., an agar‐producing red alga introduced from northeast Asia to Europe and North America, is often highly abundant in invaded areas. To assay its genetic diversity and identify the putative source of invasive populations, we analyzed the mitochondrial cytochrome c oxidase subunit I (cox1) gene from 312 individuals of G. vermiculophylla collected in 37 native and 32 introduced locations. A total of 19 haplotypes were detected: 17 in northeast Asia and three in Europe and eastern and western North America, with only one shared among all regions. The shared haplotype was present in all introduced populations and in ～99% of individuals in the introduced areas. This haplotype was also found at three native locations in east Korea, west Japan, and eastern Russia. Both haplotype and nucleotide diversities were extremely low in Europe and North America compared to northeast Asia. Our study indicates that the East Sea/Sea of Japan is a likely donor region of the invasive populations of G. vermiculophylla in the east and west Atlantic and the east Pacific.     

Growth and protection against oxidative stress in young clones and mature spruce trees (Picea abies L.) at high altitudes

Clones of Norway spruce (Picea abies L.) were grown for several years on an altitudinal gradient (1750 m, 1150 m and 800 m above sea level) to study the effects of environmental × genetic interactions on growth and foliar metabolites (protein, pigments, antioxidants). Clones at the tree line showed 4.3-fold lower growth rates and contained 60% less chlorophyll (per gram of dry matter) than those at valley level. The extent of growth reduction was clone-dependent. The mortality of the clones was low and not altitude-dependent. At valley level, but not at high altitude, needles of mature spruce trees showed lower pigment and protein concentrations than clones. In general, antioxidative systems in needles of the mature trees and young clones did not increase with increasing altitude. Needles of all trees at high altitude showed higher concentrations of dehydroascorbate than at lower altitudes, indicating higher oxidative stress. In one clone, previously identified as sensitive to acute ozone doses, this increase was significantly higher and the growth reduction was stronger than in the other genotypes. This clone also displayed a significant reduction in glutathione reductase activity at high altitude. These results suggest that induction of antioxidative systems is apparently not a general prerequisite to cope with altitude in clones whose mother plants originated from higher altitudes (about 650–1100 m above sea level, Hercycnic-Carpathian distribution area), but that the genetic constitution for maintenance of high antioxidative protection is important for stress compensation at the tree line. Received: 13 October 1998 / Accepted: 22 June 1999     

Reduced glutamine concentration improves protein production in growth-arrested CHO-DG44 and HEK-293E cells

For most cultivated mammalian cells, glutamine is an essential medium component. However, glutamine consumption results in the production of ammonia, a cytotoxic byproduct. Here we investigated the effect of glutamine reduction on recombinant protein production and ammonia accumulation in transiently transfected CHO and HEK-293E cells maintained under conditions of growth arrest. Maximum transient recombinant protein yields were observed in HEK-293E cultures without glutamine and in CHO cultures with 2 mM glutamine. The initial concentration of glutamine correlated with the level of ammonia accumulation in each culture. For both a stable CHO-derived cell line and a polyclonal population of recombinant CHO cells grown under conditions of mild hypothermia, the highest volumetric protein productivity was observed in cultures without glutamine. Here, the level of ammonia accumulation also corresponded to the initial glutamine concentration. Our data demonstrate that reduction of glutamine in the medium is an effective approach to improve protein production in both transiently and stably transfected mammalian cells when applying conditions that reduce or arrest the growth of these cells.