Blocking and detection chemistries affect antibody performance on reverse phase protein arrays

Antibody specificity is critical for RP protein arrays (RPA). The effects of blocking and detection chemistries on antibody specificity were evaluated for Western blots and RPA. Blocking buffers significantly affected nonspecific banding on Western blots, with corresponding effects on arrays. Tyramide signal amplification (TSA) increased both specific and nonspecific signals on Westerns and arrays, masking the expected gradations in signal intensity. These results suggest that consistent blocking and detection conditions should be used for antibody validation and subsequent RPA experiments.     

The ionic environment determines ribozyme cleavage rate by modulation of nucleobase pK a

Several small ribozymes employ general acid–base catalysis as a mechanism to enhance site-specific RNA cleavage, even though the functional groups on the ribonucleoside building blocks of RNA have pK_a values far removed from physiological pH. The rate of the cleavage reaction is strongly affected by the identity of the metal cation present in the reaction solution; however, the mechanism(s) by which different cations contribute to rate enhancement has not been determined. Using the Neurospora VS ribozyme, we provide evidence that different cations confer particular shifts in the apparent pK_a values of the catalytic nucleobases, which in turn determines the fraction of RNA in the protonation state competent for general acid–base catalysis at a given pH, which determines the observed rate of the cleavage reaction. Despite large differences in observed rates of cleavage in different cations, mathematical models of general acid–base catalysis indicate that k₁, the intrinsic rate of the bond-breaking step, is essentially constant irrespective of the identity of the cation(s) in the reaction solution. Thus, in contrast to models that invoke unique roles for metal ions in ribozyme chemical mechanisms, we find that most, and possibly all, of the ion-specific rate enhancement in the VS ribozyme can be explained solely by the effect of the ions on nucleobase pK_a. The inference that k₁ is essentially constant suggests a resolution of the problem of kinetic ambiguity in favor of a model in which the lower pK_a is that of the general acid and the higher pK_a is that of the general base.     

Structural and functional plasticity of dendritic spines – root or result of behavior?

Dendritic spines are multifunctional integrative units of the nervous system and are highly diverse and dynamic in nature. Both internal and external stimuli influence dendritic spine density and morphology on the order of minutes. It is clear that the structural plasticity of dendritic spines is related to changes in synaptic efficacy, learning and memory and other cognitive processes. However, it is currently unclear whether structural changes in dendritic spines are primary instigators of changes in specific behaviors, a consequence of behavioral changes, or both. In this review, we first examine the basic structure and function of dendritic spines in the brain, as well as laboratory methods to characterize and quantify morphological changes in dendritic spines. We then discuss the existing literature on the temporal and functional relationship between changes in dendritic spines in specific brain regions and changes in specific behaviors mediated by those regions. Although technological advancements have allowed us to better understand the functional relevance of structural changes in dendritic spines that are influenced by environmental stimuli, the role of spine dynamics as an underlying driver or consequence of behavior still remains elusive. We conclude that while it is likely that structural changes in dendritic spines are both instigators and results of behavioral changes, improved research tools and methods are needed to experimentally and directly manipulate spine dynamics in order to more empirically delineate the relationship between spine structure and behavior.     

Comparison of diet and feeding activity of two polychaetes, Nephtys caeca (Fabricius) and Nereis virens (Sars), in an estuarine intertidal environment in Québec, Canada

It is crucial to study how food resources are allocated in order to understand how invertebrate species of soft-bottomed benthic communities can live in the same environment, especially when belonging to the same trophic level. On the southern shore of the Lower St. Lawrence Estuary, the polychaetes Nereis virens (Nereidae) and Nephtys caeca (Nephtyidae) represent the main infaunal predators and are known to regulate (by predation) other populations within their community. For these animal populations, there is a high degree of spatial overlap in the horizontal dimension as well as vertically in the sediment column. The purpose of this study was to describe and compare the feeding activity of N. virens and N. caeca in relation to several environmental factors. The feeding activity of N. caeca was further studied under natural and experimental conditions and a feeding experiment was carried out to evaluate how the food resource is subdivided in conditions of high spatial overlap. Finally, the prospecting activity of N. virens was described under natural conditions. N. virens and N. caeca exhibited a high degree of dietary overlap, particularly at the adult stage. This food resource overlap can easily influence the regulation of these species, even though they display different feeding responses to environmental stimuli. Tides and storm events can also influence the feeding behaviour. We conclude that different behavioural feeding responses likely favour the occurrence of these two species in the same trophic environment.     

A lipophilic adjuvant carrier system for antigenic peptides

A lipoamino acid based synthetic peptide, (Lipid Core Peptide, LCP) derived from the conserved region of group A streptococci (GAS) was evaluated as potential candidate in a vaccine to prevent GAS-associated diseases, including rheumatic heart disease and post-streptococcal acute glomerulonephritis. Multiple copies of a peptide sequence from the bacterial surface M protein were incorporated into a lipid core and it was used to immunize mice with and without the application of adjuvant. The LCP construct had significantly enhanced immunogenicity compared with the monomeric peptide epitope. Furthermore, the peptides incorporated into the LCP system generated antibodies without the use of any conventional adjuvant.     

A Preliminary Approach to the Use of the Isotopic Ratio 13C/12C for the Evaluation of Mineralization in Aquatic Environments

¹²C/¹³C ratio is used as natural tracer for the estimation of the mineralizing activity of pelagic bacteria. Attention is particularly given to the variations of the isotopic ratio of the inorganic carbon produced by the microbial oxidation in comparison with the ratio of the dissolved organic matter.     

Lipid and carbohydrate based adjuvant/carriers in immunology.

This review discusses various issues regarding vaccines; what are they and how they work, safety aspects, the role of adjuvants and carriers in vaccination, synthetic peptides as immunogens, and new technologies for vaccine development and delivery including the identification of novel adjuvants for mucosal vaccine delivery. There has been a recent increase of interest in the use of lipids and carbohydrates as adjuvants, and so a particular emphasis is placed on adjuvants derived from lipids or carbohydrates, or from both.     

Mutant p53 gain of function in two mouse models of Li-Fraumeni syndrome

The p53 tumor suppressor gene is commonly altered in human tumors, predominantly through missense mutations that result in accumulation of mutant p53 protein. These mutations may confer dominant-negative or gain-of-function properties to p53. To ascertain the physiological effects of p53 point mutation, the structural mutant p53R172H and the contact mutant p53R270H (codons 175 and 273 in humans) were engineered into the endogenous p53 locus in mice. p53R270H/+ and p53R172H/+ mice are models of Li-Fraumeni Syndrome; they developed allele-specific tumor spectra distinct from p53+/- mice. In addition, p53R270H/- and p53R172H/- mice developed novel tumors compared to p53-/- mice, including a variety of carcinomas and more frequent endothelial tumors. Dominant effects that varied by allele and function were observed in primary cells derived from p53R270H/+ and p53R172H/+ mice. These results demonstrate that point mutant p53 alleles expressed under physiological control have enhanced oncogenic potential beyond the simple loss of p53 function.     

2-ethoxycarbonyl-2-methyl-3,4-dihydro-2H-pyrrole-1-oxide: evaluation of the spin trapping properties

The 2-ethoxycarbonyl-2-methyl-3,4-dihydro-2H-pyrrole-l-oxide (EMPO), an easily prepared pyrroline-N-oxide has been tested as a free radical scavenger. Spin adducts of superoxide, hydroxyl radical, and other free radicals were characterized in phosphate buffer at pH 7.0 and 5.6. At pH 7 in phosphate buffer, the EMPO/O(2)(-*) spin adduct was estimated to be about five times more persistent than its DMPO analogue. Furthermore, its decay does not produce the EMPO/HO&z.rad; adduct.