Lack of hypoxic stimulation of VEGF secretion from neutrophils and platelets

Low oxygen (O(2)) is the key stimulus for expression of vascular endothelial growth factor (VEGF) in several adherent cells. Whether hypoxia also directs the release of VEGF protein from neutrophils (polymorphonuclear neutrophils; PMN) and platelets has not been investigated. We therefore compared VEGF release of platelets, PMN, and human vascular smooth muscle cells (HSMC) in response to hypoxia with that to activators of cellular degranulation. In contrast to HSMC, VEGF release from PMN and platelets or VEGF mRNA expression in PMN was not stimulated under hypoxic conditions (1% O(2)). Hypo- or hyperthermia and acidosis, other conditions potentially associated with ischemic and inflammatory tissue injury, also did not stimulate VEGF secretion from PMN. However, stimulation of platelets with thrombin and of PMN with phorbol 12-myristate 13-acetate induced a time-dependent release of VEGF, peaking after 30 and 60 min, respectively. This was blocked by the degranulation inhibitor pentoxifylline but not by the protein-synthesis inhibitor cycloheximide. We conclude that rapid release of VEGF from platelets and PMN may occur independently of oxygenation during inflammation and hemostasis.     

Dynamics and ordering in a spin-labeled oligonucleotide observed by 220 GHz electron paramagnetic resonance

The dynamics of a newly synthesized cytosine spin-label and the spin-labeled pentamer TTC*TT have been observed by high-frequency (220 GHz) electron paramagnetic resonance (EPR) in aqueous solution at ambient temperature using only nanomolar amounts of spin-label. Temperature studies were carried out for both labeled species in buffer containing glycerol. The motion of the spin-labeled monomer could be fitted using a model of fully anisotropic rotation (FAR) over the entire temperature range studied. In the single-stranded pentamer, the high-field spectra are best interpreted using a model of microscopic ordering with macroscopic disorder (MOMD) with the probe in a highly nonpolar environment. The observed local order parameters of 0.60-0.70 suggest a micelle-like structure in which the label is tightly packed with the hydrophobic bases. These preliminary studies illustrate how the excellent orientation selectivity of high-field EPR provides new dynamic information about local base motions in DNA, and also how high-field EPR of spin-labels allows one to discriminate accurately between the effects of local versus global motions in spin-labeled macromolecules.     

Chimeric yellow fever/dengue virus as a candidate dengue vaccine: quantitation of the dengue virus-specific CD8 T-cell response

We have constructed a chimeric yellow fever/dengue (YF/DEN) virus, which expresses the premembrane (prM) and envelope (E) genes from DEN type 2 (DEN-2) virus in a YF virus (YFV-17D) genetic background. Immunization of BALB/c mice with this chimeric virus induced a CD8 T-cell response specific for the DEN-2 virus prM and E proteins. This response protected YF/DEN virus-immunized mice against lethal dengue encephalitis. Control mice immunized with the parental YFV-17D were not protected against DEN-2 virus challenge, indicating that protection was mediated by the DEN-2 virus prM- and E-specific immune responses. YF/DEN vaccine-primed CD8 T cells expanded and were efficiently recruited into the central nervous systems of DEN-2 virus challenged mice. At 5 days after challenge, 3 to 4% of CD8 T cells in the spleen were specific for the prM and E proteins, and 34% of CD8 T cells in the central nervous system recognized these proteins. Depletion of either CD4 or CD8 T cells, or both, strongly reduced the protective efficacy of the YF/DEN virus, stressing the key role of the antiviral T-cell response.     

Halorespiring bacteria-molecular characterization and detection

Recently, a rapidly increasing number of bacteria has been isolated that is able to couple the reductive dehalogenation of various halogenated aromatic and aliphatic compounds like chlorophenols and tetrachloroethene to energy conservation by electron-transport-coupled phosphorylation. The potential of these halorespiring bacteria for innovative clean-up strategies of polluted anoxic environments has greatly stimulated efforts to unravel the molecular basis of the novel respiratory chains they possess. The thorough characterization of halorespiratory key components at the physiological, biochemical and molecular genetic level has revealed both structural and functional similarity of chloroaryl- and chloroalkyl-respiratory chains from different phylogenetically distinct microorganisms. The reductive dehalogenases from halorespiring bacteria were found to comprise a novel class of corrinoid-containing Fe/S-proteins. Sensitive molecular methods for monitoring both presence and fate of halorespiring bacteria have been developed, which will be instrumental for the design and maintenance of optimised in situ bioremediation processes.     

Plant endemism in two forests in southern Bahia, Brazil

An important factor in determining species rarity is the geographic distribution of species. Estimates were made of the level of endemism of the flora of two sites in the southern Bahian wet forest zone. Estimates were made for endemism in the Atlantic forest biome and for the much more restricted area of southern Bahia and northern Espi´rito Santo and are derived from analyses of the distributions of the species known from each area. The species checklist for each area is based on identified specimens resulting from intensive collecting in a forest near Serra Grande (40km north of Ilhe´us) and the Una Biological Reserve (40km south of Ilhe´us). Slightly less than half of the species (45.2% at Una and 47.7% at Serra Grande) have widespread distributions and 7.4% at each site are disjunct between the coastal forests and Amazonia. In the Una Reserve, 44.1% of the species are endemic to the coastal forest and 28.1% endemic to southern Bahia and northern Espi´rito Santo. At Serra Grande, 41.6% of the species are endemic to the coastal forest and 26.5% endemic to southern Bahia and northern Espi´rito Santo.     

Understanding the mechanism of virus removal by Q sepharose fast flow chromatography during the purification of CHO‐cell derived biotherapeutics

During production of therapeutic monoclonal antibodies (mAbs) in mammalian cell culture, it is important to ensure that viral impurities and potential viral contaminants will be removed during downstream purification. Anion exchange chromatography provides a high degree of virus removal from mAb feedstocks, but the mechanism by which this is achieved has not been characterized. In this work, we have investigated the binding of three viruses to Q sepharose fast flow (QSFF) resin to determine the degree to which electrostatic interactions are responsible for viral clearance by this process. We first used a chromatofocusing technique to determine the isoelectric points of the viruses and established that they are negatively charged under standard QSFF conditions. We then determined that virus removal by this chromatography resin is strongly disrupted by the presence of high salt concentrations or by the absence of the positively charged Q ligand, indicating that binding of the virus to the resin is primarily due to electrostatic forces, and that any non‐electrostatic interactions which may be present are not sufficient to provide virus removal. Finally, we determined the binding profile of a virus in a QSFF column after a viral clearance process. These data indicate that virus particles generally behave similarly to proteins, but they also illustrate the high degree of performance necessary to achieve several logs of virus reduction. Overall, this mechanistic understanding of an important viral clearance process provides the foundation for the development of science‐based process validation strategies to ensure viral safety of biotechnology products. Biotechnol. Bioeng. 2009; 104: 371–380 © 2009 Wiley Periodicals, Inc.     

Comment on M. Guillot‐Delost et al. (2008;10:834‐846): Clinical‐grade preparation of human natural regulatory T cells encoding the thymidine kinase suicide gene as a safety gene

This Letter to the Editor has a published Response, which can be viewed using the link below. Response to Comment     

Spag17 Deficiency Results in Skeletal Malformations and Bone Abnormalities

Height is the result of many growth and development processes. Most of the genes associated with height are known to play a role in skeletal development. Single-nucleotide polymorphisms in the SPAG17 gene have been associated with human height. However, it is not clear how this gene influences linear growth. Here we show that a targeted mutation in Spag17 leads to skeletal malformations. Hind limb length in mutants was significantly shorter than in wild-type mice. Studies revealed differences in maturation of femur and tibia suggesting alterations in limb patterning. Morphometric studies showed increased bone formation evidenced by increased trabecular bone area and the ratio of bone area to total area, leading to reductions in the ratio of marrow area/total area in the femur. Micro-CTs and von Kossa staining demonstrated increased mineral in the femur. Moreover, osteocalcin and osterix were more highly expressed in mutant mice than in wild-type mice femurs. These data suggest that femur bone shortening may be due to premature ossification. On the other hand, tibias appear to be shorter due to a delay in cartilage and bone development. Morphometric studies showed reduction in growth plate and bone formation. These defects did not affect bone mineralization, although the volume of primary bone and levels of osteocalcin and osterix were higher. Other skeletal malformations were observed including fused sternebrae, reduced mineralization in the skull, medial and metacarpal phalanges. Primary cilia from chondrocytes, osteoblasts, and embryonic fibroblasts (MEFs) isolated from knockout mice were shorter and fewer cells had primary cilia in comparison to cells from wild-type mice. In addition, Spag17 knockdown in wild-type MEFs by Spag17 siRNA duplex reproduced the shorter primary cilia phenotype. Our findings disclosed unexpected functions for Spag17 in the regulation of skeletal growth and mineralization, perhaps because of its role in primary cilia of chondrocytes and osteoblasts.     

Neural Correlates of Emotional Interference in Social Anxiety Disorder

Disorder-relevant but task-unrelated stimuli impair cognitive performance in social anxiety disorder (SAD); however, time course and neural correlates of emotional interference are unknown. The present study investigated time course and neural basis of emotional interference in SAD using event-related functional magnetic resonance imaging (fMRI). Patients with SAD and healthy controls performed an emotional stroop task which allowed examining interference effects on the current and the succeeding trial. Reaction time data showed an emotional interference effect in the current trial, but not the succeeding trial, specifically in SAD. FMRI data showed greater activation in the left amygdala, bilateral insula, medial prefrontal cortex (mPFC), dorsal anterior cingulate cortex (ACC), and left opercular part of the inferior frontal gyrus during emotional interference of the current trial in SAD patients. Furthermore, we found a positive correlation between patients’ interference scores and activation in the mPFC, dorsal ACC and left angular/supramarginal gyrus. Taken together, results indicate a network of brain regions comprising amygdala, insula, mPFC, ACC, and areas strongly involved in language processing during the processing of task-unrelated threat in SAD. However, specifically the activation in mPFC, dorsal ACC, and left angular/supramarginal gyrus is associated with the strength of the interference effect, suggesting a cognitive network model of attentional bias in SAD. This probably comprises exceeded allocation of attentional resources to disorder-related information of the presented stimuli and increased self-referential and semantic processing of threat words in SAD.