A combination of intradermal jet-injection and electroporation overcomes in vivodose restriction of DNA vaccines

Development of various vaccines for prostate cancer (PCa) is becoming an active research area. PCa vaccines are perceived to have less toxicity compared with the available cytotoxic agents. While various immune-based strategies can elicit anti-tumour responses, DNA vaccines present increased efficacy, inducing both humoural and cellular immunity. This immune activation has been proven effective in animal models and initial clinical trials are encouraging. However, to validate the role of DNA vaccination in currently available PCa management paradigms, strong clinical evidence is still lacking. This article provides an overview of the basic principles of DNA vaccines and aims to provide a summary of preclinical and clinical trials outlining the benefits of this immunotherapy in the management of PCa.     

Stimulus dependency of object-evoked responses in human visual cortex: an inverse problem for category specificity

Many studies have linked the processing of different object categories to specific event-related potentials (ERPs) such as the face-specific N170. Despite reports showing that object-related ERPs are influenced by visual stimulus features, there is consensus that these components primarily reflect categorical aspects of the stimuli. Here, we re-investigated this idea by systematically measuring the effects of visual feature manipulations on ERP responses elicited by both structure-from-motion (SFM)-defined and luminance-defined object stimuli. SFM objects elicited a novel component at 200-250 ms (N250) over parietal and posterior temporal sites. We found, however, that the N250 amplitude was unaffected by restructuring SFM stimuli into meaningless objects based on identical visual cues. This suggests that this N250 peak was not uniquely linked to categorical aspects of the objects, but is strongly determined by visual stimulus features. We provide strong support for this hypothesis by parametrically manipulating the depth range of both SFM- and luminance-defined object stimuli and showing that the N250 evoked by SFM stimuli as well as the well-known N170 to static faces were sensitive to this manipulation. Importantly, this effect could not be attributed to compromised object categorization in low depth stimuli, confirming a strong impact of visual stimulus features on object-related ERP signals. As ERP components linked with visual categorical object perception are likely determined by multiple stimulus features, this creates an interesting inverse problem when deriving specific perceptual processes from variations in ERP components.     

Peptide-binding GRP78 protects neurons from hypoxia-induced apoptosis

Brain ischemia has major consequences leading to the apoptosis of astrocytes and neurons. Glucose-regulated protein 78 (GRP78) known for its role in endoplasmic reticulum stress alleviation was discovered on several cell surfaces acting as a receptor for signaling pathways. We have previously described peptides that bind cell surface GRP78 on endothelial cells to induce angiogenesis. We have also reported that ADoPep1 binds cardiomyocytes to prevent apoptosis of ischemic heart cells. In this study we describe the effect of hypoxia on astrocytes and neurons cell surface GRP78. Under hypoxic conditions, there was an increase of more than fivefold in GRP78 on cell surface of neurons while astrocytes were not affected. The addition of the GRP78 binding peptide, ADoPep1, to neurons decreased the percentage of GRP78 positive cells and did not change the percent of astrocytes. However, a significant increase in early and late apoptosis of both astrocytes and neurons under hypoxia was attenuated in the presence of ADoPep1. Intravitreal administration of ADoPep1 to mice in a model of optic nerve crush significantly reduced retinal cell loss after 21 days compared to the crush-damaged eyes without treatment or by control saline vehicle injection. Histological staining demonstrated reduced GRP78 after ADoPep1 treatment. The mechanism of peptide neuroprotection was demonstrated by the inhibition of hypoxia induced caspase 3/7 activity, cytochrome c release and p38 phosphorylation. This study is the first report on hypoxic neuronal and astrocyte cell surface GRP78 and suggests a potential therapeutic target for neuroprotection.     

Distinct role of subcomplexes of the COPI coat in the regulation of ArfGAP2 activity

COPI vesicles serve for transport of proteins and membrane lipids in the early secretory pathway. Their coat protein (coatomer) is a heptameric complex that is recruited to the Golgi by the small GTPase Arf1. Although recruited en bloc, coatomer can be viewed as a stable assembly of an adaptin‐like tetrameric subcomplex (CM4) and a trimeric ‘cage’ subcomplex (CM3). Following recruitment, coatomer stimulates ArfGAP‐dependent GTP hydrolysis on Arf1. Here, we employed recombinant coatomer subcomplexes to study the role of coatomer components in the regulation of ArfGAP2, an ArfGAP whose activity is strictly coatomer‐dependent. Within CM4, we define a novel hydrophobic pocket for ArfGAP2 interaction on the appendage domain of γ₁‐COP. The CM4 subcomplex (but not CM3) is recruited to membranes through Arf1 and can subsequently recruit ArfGAP2. Neither CM3 nor CM4 in itself is effective in stimulating ArfGAP2 activity, but stimulation is regained when both subcomplexes are present. Our findings point to a distinct role of each of the two coatomer subcomplexes in the regulation of ArfGAP2‐dependent GTP hydrolysis on Arf1, where the CM4 subcomplex functions in GAP recruitment, while, similarly to the COPII system, the cage‐like CM3 subcomplex stimulates the catalytic reaction.     

Formation of Phosphoglycosides in Caenorhabditis elegans: A Novel Biotransformation Pathway

Background

Caenorhabditis elegans (C. elegans) has become a widely used model to explore the effect of food constituents on health as well as on life-span extension. The results imply that besides essential nutrients several flavonoids are able to impact the aging process. What is less investigated is the bioavailability and biotransformation of these compounds in C. elegans. In the present study, we focused on the soy isoflavone genistein and its metabolism in the nematode as a basis for assessing whether this model system mimics the mammalian condition.

Principal Findings

C. elegans was exposed to 100 µM genistein for 48 hours. The worm homogenate was extracted and analyzed by liquid chromatography (LC). 11 metabolites of genistein were detected and characterized using LC electrospray ionization mass spectrometry. All genistein metabolites formed by C. elegans were found to be sugar conjugates, primarily genistein-O-glucosides. The dominant metabolite was identified as genistein-7-O-phosphoglucoside. Further interesting metabolites include two genistein-di-O-glycosides, a genistein-O-disaccharide as well as a genistein-O-phosphodisaccharide.

Conclusions/Significance

Our study provides evidence for a novel biotransformation pathway in C. elegans leading to conjugative metabolites which are not known for mammals. The metabolism of genistein in mammals and in C. elegans differs widely which may greatly impact the bioactivity. These differences need to be appropriately taken into consideration when C. elegans is used as a model to assess possible health or aging effects.     

The paramutated SULFUREA locus of tomato is involved in auxin biosynthesis

The tomato (Solanum lycopersicum) sulfurea mutation displays trans-inactivation of wild-type alleles in heterozygous plants, a phenomenon referred to as paramutation. Homozygous mutant plants and paramutated leaf tissue of heterozygous plants show a pigment-deficient phenotype. The molecular basis of this phenotype and the function of the SULFUREA gene (SULF) are unknown. Here, a comprehensive physiological analysis of the sulfurea mutant is reported which suggests a molecular function for the SULFUREA locus. It is found that the sulf mutant is auxin-deficient and that the pigment-deficient phenotype is likely to represent only a secondary consequence of the auxin deficiency. This is most strongly supported by the isolation of a suppressor mutant which shows an auxin overaccumulation phenotype and contains elevated levels of indole-3-acetic acid (IAA). Several lines of evidence point to a role of the SULF gene in tryptophan-independent auxin biosynthesis, a pathway whose biochemistry and enzymology is still completely unknown. Thus, the sulfurea mutant may provide a promising entry point into elucidating the tryptophan-independent pathway of IAA synthesis.     

Regulatory mechanisms underlying agmatine homeostasis in humans

Regulation of agmatine homeostasis has so far only been poorly defined. In the present study, three mechanisms regulating human agmatine homeostasis were investigated. 1) Enzymatic regulation: expression of arginine decarboxylase, diamine oxidase, and ornithine decarboxylase in human colon neoplastic tissue was, at the mRNA level, about 75% and 50% lower and 150% higher, respectively, than in the adjacent normal tissue; expression of agmatinase was unchanged. 2) Bacteria-derived agmatine: ten representative bacteria strains of the human intestinal microbiota considerably differed in agmatine production and its efflux into their surrounding fluid, suggesting that the composition of the intestinal microbiota influences the agmatine availability in the gut lumen for absorption. 3) Regulation of blood plasma agmatine concentration by the human liver: at low concentrations in portal venous blood plasma, agmatine either slightly increased or further decreased in blood plasma through liver passage. Above a threshold of 14 ng/ml agmatine in the portal venous blood plasma, substantial hepatic agmatine removal from blood occurred. Taken together, a perturbation of agmatine homeostasis has been proven to be involved in the regulation of malignant cell proliferation. The amount of agmatine available for absorption, which is an important physiological source of agmatine in the human organism, should differ considerably depending on the composition of the bacterial flora in the chyme since the various species of intestinal bacteria largely differ in their ability to form agmatine. Finally, evidence has been presented that the liver plays a crucial physiological role in the maintenance of agmatine homeostasis in the human organism.