Two‐dimensional reference map for the basic proteome of the human dorsolateral prefrontal cortex (dlPFC) of the prefrontal lobe region of the brain

We describe a 2‐DE proteomic reference map containing 227 basic proteins in the dorsolateral prefrontal cortex region of the human brain. Proteins were separated in the first dimension on pH 6–11 IPG strips using paper‐bridge loading and on 12% SDS‐PAGE in the second dimension. Proteins were subsequently identified by MS and spectra were analyzed using an in‐house proteomics data analysis platform, Proline. The 2‐DE reference map is available via the UCD 2‐DE Proteome Database ( http://proteomics‐portal.ucd.ie:8082 ) and can also be accessed via the WORLD‐2DPAGE Portal ( http://www.expasy.ch/world‐2dpage/ ). The associated protein identification data have been submitted to the PRIDE database (accession numbers 10018–10033). Separation of proteins in the basic region resolves more membrane associated proteins relevant to the synaptic pathology central to many neurological disorders. The 2‐DE reference map will aid with further characterisation of neurological disorders such as bipolar and schizophrenia.     

Utilization of trehalose during Dictyostelium discoideum spore germination

An analysis of metabolism by measurement of respiratory quotient values indicates that reduced substances, such as lipids and/or amino acids, are the primary respiratory substrates of dormant Dictyostelium discoideum spores. The spores appear to consume both reduced substances and carbohydrates during the swelling stage of germination. The respiration of emerged myxamoebae is again dominated by the consumption of reduced substances. The pool of trehalose remains largely intact during heat-induced activation and also during postactivation lag. The initiation of spore swelling is accompanied by a decrease in the trehalose pool; the majority of trehalose is consumed before late spore swelling. Upon placing heat-activated spores under restrictive environmental conditions, swelling and trehalose hydrolysis are both prevented. Release from these conditions results in rapid swelling and hydrolysis of trehalose. Trehalase, the enzyme responsible for trehalose breakdown, is present in dormant spores at basal levels. This preformed enzyme is responsible for the hydrolysis of trehalose even though there is a significant increase in trehalase activity with the emergence of myxamoebae. RNA and protein synthesis inhibitors do not prevent trehalose hydrolysis or spore swelling. It is concluded that oxidation of reduced substances occurs in dormant, activated, and swollen spores, as well as in emerged myxamoebae of D. discoideum. Carbohydrate utilization dominates over the oxidation of reduced substances only during the swelling stage of germination.     

Differential roles of ERK1/2 and JNK in retinal development and degeneration

Programmed cell death is well established as a key factor in the development of the vertebrate nervous system of which the retina is a unique sensory component. However, it is of utmost importance for the survival of post-mitotic tissues such as the retina that the execution of the cell death program is kept under stringent control once development is complete. This is exemplified by the many retinal dystrophies where aberrant apoptosis results in loss of distinct cell layers in the mature retina and often culminates in blindness. In this study, we report that the extracellular signal-regulated kinase (ERK1/2) pathway plays a key role in the regulation of apoptosis during retinal development. We show that as the retina matures, the emphasis shifts towards survival and ERK1/2 is activated resulting in phosphorylation of the potent BH3-only protein Bim(EL) and a dramatic decline in Bim(EL) expression via proteasomal degradation. We find that activation of ERK1/2 also occurs in response to injury in retinal explants. However, this is a transient response and appears to be overcome by Jun N-terminal kinase activation resulting in induction of Bim(EL) mRNA and photoreceptor apoptosis. Our findings provide new insights into the intracellular pathways responsible for regulating apoptosis during neuronal development and degeneration.     

Insertional Mutagenesis To Generate Lantibiotic Resistance in Lactococcus lactis

While the potential emergence of food spoilage and pathogenic bacteria with resistance to lantibiotics is a concern, the creation of derivatives of starter cultures and adjuncts that can grow in the presence of these antimicrobials may have applications in food fermentations. Here a bank of Lactococcus lactis IL1403 mutants was created and screened, and a number of novel genetic loci involved in lantibiotic resistance were identified.     

The ability to cleave 28S ribosomal RNA during apoptosis is a cell-type dependent trait unrelated to DNA fragmentation

Discrete cleavages within 28S rRNA divergent domains have previously been found to coincide with DNA fragmentation during apoptosis. Here we show that rRNA and DNA cleavages can occur independently in apoptotic cells, i.e. that the previously observed correlation is likely to be coincidental. In HL-60 cells, apoptosis with massive DNA fragmentation could be induced without any signs of rRNA cleavage. The opposite situation; rRNA cleavage without concomitant internucleosomal DNA fragmentation, was found in okadaic acid-treated Molt-4 cells. Other leukemia cell lines underwent apoptosis either without (K562 and Molt-3) or with (U937) both forms of polynucleotide cleavage. In K562 cells transfected with a temperature-sensitive p53 mutant, internucleosomal DNA fragmentation but not 28S rRNA cleavage was inducible by wild-type p53 expression. The absence of apoptotic rRNA cleavage in some cell types suggests that this phenomenon is tightly regulated and unrelated to DNA fragmentation or a presumed scheme for general macromolecular degradation in apoptotic cells.     

Immunohistochemistry in the evaluation of neovascularization in tumor xenografts

Angiogenesis, or neovascularization, is known to play an important role in the neoplastic progression leading to metastasis. CD31 or Factor VIII-related antigen (F VIII RAg) immunohistochemistry is widely used in experimental studies for quantifying tumor neovascularization in immunocompromised animal models implanted with transformed human cell lines. Quantification, however, can be affected by variations in the methodology used to measure vascularization including antibody selection, antigen retrieval (AR) pretreatment, and evaluation techniques. To examine this further, we investigated the microvessel density (MVD) and the intensity of microvascular staining among five different human tumor xenografts and a mouse syngeneic tumor using anti-CD31 and F VIII RAg immunohistochemical staining. Different AR methods also were evaluated. Maximal retrieval of CD31 was achieved using 0.5 M Tris (pH 10) buffer, while maximum retrieval of F VIII RAg was achieved using 0.05% pepsin treatment of tissue sections. For each optimized retrieval condition, anti-CD31 highlighted small vessels better than F VIII RAg. Furthermore, the MVD of CD31 was significantly greater than that of F VIII RAg decorated vessels (p<0.001). The choice of antibody and AR method has a significant affect on immunohistochemical findings when studying angiogenesis. One also must use caution when comparing studies in the literature that use different techniques and reagents.     

Morphological and molecular characterization of populations of plains bristlegrass (<i>Setaria macrostachya</i> Kunth) in Chihuahua,México

Plains bristlegrass (Setaria macrostachya Kunth) is a native grass with forage value. However, due to the lack of grazing management practices, populations and thus genetic diversity, have been reduced. Morphological and genetic variability were analyzed on 44 populations of plains bristlegrass in the State of Chihuahua. Plants were transplanted in a common area under natural conditions. Two years later, morphological characterization was evaluated measuring nine variables, and genetic variability using AFLP molecular markers. The principal components analysis (PC) showed that the three first principal components explained 73.74% of the variation. The variables with the greatest contribution to the variance in PC1 were plant height and inflorescence length; in CP2, tiller number and leaf width; and in PC3, tiller thickness. Application of four pairs of primers, presented 186 total bands, from which 87.10% showed polymorphism and 12.90% monomorphism. The combination of EcoRI-AGG MseI-CAG primers detected the highest percentage (93%) of polymorphism with 40 polymorphic bands. The cluster analysis and Dice coefficient indicated that populations clump into two groups. The wide genetic variability and morphological characteristics detected among populations represent the basis for the selection of populations that could be used with different purposes in the rehabilitation of ecosystems. In addition, this study will allow establishment of in situ conservation strategies.     

DNA sequence has an effect on the extent and kinds of alkylation of DNA by a potent carcinogen

A system has been developed to study the effects of base sequence (neighboring bases) upon the alkylation of guanine (G) and adenine (A) bases in DNA. The study was performed on the synthetic polydeoxyribonucleotides, poly(dG).poly(dC), poly(dG-dC).poly(dG-dC), poly(dA).poly(dT), poly(dA-dT).poly(dA-dT), poly(dA-dC).poly(dG-dT), poly(dA-dG).poly(dC-dT), as well as calf thymus DNA. Each polynucleotide was treated with N-[3H]methyl-N-nitrosourea (MNU), depurinated, and the freed alkylpurines separated by HPLC and quantitated by liquid scintillation counting. The amounts of 3-methylguanine (3-MG), 7-MG, and O6-MG relative to guanine, and 3-methyladenine (3-MA) and 1-MA plus 7-MA relative to adenine, and also the O6-MG/7-MG ratios were highly reproducible for a given polynucleotide. Significant differences were found in the amounts of each of the methylpurines formed when compared among the six synthetic polynucleotides and DNA. This evidence is interpreted as an effect upon alkylation which is ultimately dependent upon the base sequence. These findings may have significance in defining the specificity of chemical carcinogens in terms of the susceptability to modification of nucleotide sequences such as those found in certain oncogenes.     

Stress-induced activation of Nox contributes to cell survival signalling via production of hydrogen peroxide

Reactive oxygen species (ROS) have traditionally been viewed as a toxic group of molecules; however, recent publications have shown that these molecules, including H₂O₂, can also strongly promote cell survival. Even though the retina has a large capacity to produce ROS, little is known about its non-mitochondrial sources of these molecules, in particular the expression and function of NADPH oxidase (Nox) proteins which are involved in the direct generation of superoxide and indirectly H₂O₂. This study demonstrated that 661W cells, a retina-derived cell line, and mouse retinal explants express Nox2, Nox4 and certain of their well-established regulators. The roles of Nox2 and Nox4 in producing pro-survival H₂O₂ were determined using 661W cells and some of the controlling factors were identified. To ascertain if this phenomenon could have physiological relevance, the novel technique of time-lapse imaging of dichlorofluorescein fluorescence (generated upon H₂O₂ production) in retinal explants was established and it showed that explants also produce a burst of H₂O₂. The increase in H₂O₂ production was partly blocked by an inhibitor of Nox proteins. Overall, this study demonstrates a pro-survival role of Nox2 and Nox4 in retina-derived cells, elucidates some of the regulatory mechanisms and reveals that a similar phenomenon exists in retinal tissue as a whole.     

Circularly polarized luminescence of Sm (III) and Eu (III) complexes with chiral ligand (R/S)‐BINAPO

Luminescent lanthanide (III) ions have been exploited for circularly polarized luminescence (CPL) for decades. However, very few of these studies have involved chiral samarium (III) complexes. Complexes are prepared by mixing axial chiral ligands (R/S))‐2,2’‐bis(diphenylphosphoryl)‐1,1′‐binaphthyl (BINAPO) with europium and samarium Tris (trifluoromethane sulfonate) (Eu (OTf)₃ and Sm (OTf)₃). Luminescence‐based titration shows that the complex formed is Ln((R/S)‐BINAPO)₂(OTf)₃, where Ln = Eu or Sm. The CPL spectra are reported for Eu((R/S)‐BINAPO)₂(OTf)₃ and Sm((R/S)‐BINAPO)₂(OTf)₃. The sign of the dissymmetry factors, g_em, was dependent upon the chirality of the BINAPO ligand, and the magnitudes were relatively large. Of all of the complexes in this study, Sm((S)‐BINAPO)₂(OTf)₃ has the largest g_em = 0.272, which is one of the largest recorded for a chiral Sm³⁺ complex. A theoretical three‐dimensional structural model of the complex that is consistent with the experimental observations is developed and refined. This report also shows that (R/S)‐BINAPO are the only reported ligands where g_em (Sm³⁺) > g_em (Eu³⁺).