Characterization of the pH-mediated solubility of Bacillus thuringiensis var. san diego native delta-endotoxin crystals.

Native crystals of Bacillus thuringiensis var. san diego, a coleopteran-specific delta-endotoxin, were metabolically labelled with [35S]methionine. Specific activity was 82,000 CPM/micrograms (2.44 Ci/mmol). Using a universal buffer formulated with the same ionic strength at every pH, we determined that native crystals dissolve above pH 10 and below pH 4. At the acidic pH, the rate of solubilization was substantially slower than at the alkaline pH. Recrystallization rates for the toxin were similar regardless of solubilization conditions. The banding patterns in denatured polyacrylamide gel electrophoresis were unaffected by solubilization conditions. Toxicity was higher for soluble toxin compared to crystal toxin, but virtually identical for the acidic and alkaline produced solutions. Acid solubilization is significant because of the acidic midgut of susceptible Coleoptera.     

The utility of wing morphometrics for assigning type specimens to cryptic bumblebee species

Since the beginning of taxonomy, species have been described based on morphology, but the advent of using semio-chemicals and genetics has led to the discovery of cryptic species (i.e. morphologically similar species). When a new cryptic species is described, earlier type specimens have to be re-evaluated, although this process can be challenging as only nondestructive methods ought to be used in order to preserve the integrity of the type specimens. Methods should allow comparison with recently collected specimens clustered based on chemical, ethological and/or genetic traits with old specimens (i.e. type specimens) where only morphological traits are available. Here we develop a method based on geometric morphometric analyses of wing shape for a taxonomically challenging group of bumblebees, the subgenus Alpinobombus Skorikov. We consider nine monophyletic taxa (including several cryptic species) to assess the accuracy of this method to discriminate the taxa based on their wing shape and then to attribute type specimens using a leave-one-out cross-validation procedure. We show that, for these bees, wing shape is taxon-specific, except for two sister taxa for which the species status is still debated. Moreover, for most of the taxa, type specimens were correctly attributed with high posterior probabilities of attribution, except for a few type specimens corresponding to the same two sister taxa where taxa delimitation based on wing shape was previously the subject of discussion. Our study highlights the potential of geometric morphometric analyses to help in the re-attribution of type specimens when the existence of cryptic species is revealed.     

Recurrent Genomic Gains in Preinvasive Lesions as a Biomarker of Risk for Lung Cancer

Lung carcinoma development is accompanied by field changes that may have diagnostic significance. We have previously shown the importance of chromosomal aneusomy in lung cancer progression. Here, we tested whether genomic gains in six specific loci, TP63 on 3q28, EGFR on 7p12, MYC on 8q24, 5p15.2, and centromeric regions for chromosomes 3 (CEP3) and 6 (CEP6), may provide further value in the prediction of lung cancer. Bronchial biopsy specimens were obtained by LIFE bronchoscopy from 70 subjects (27 with prevalent lung cancers and 43 individuals without lung cancer). Twenty six biopsies were read as moderate dysplasia, 21 as severe dysplasia and 23 as carcinoma in situ (CIS). Four-micron paraffin sections were submitted to a 4-target FISH assay (LAVysion, Abbott Molecular) and reprobed for TP63 and CEP 3 sequences. Spot counts were obtained in 30–50 nuclei per specimen for each probe. Increased gene copy number in 4 of the 6 probes was associated with increased risk of being diagnosed with lung cancer both in unadjusted analyses (odds ratio = 11, p<0.05) and adjusted for histology grade (odds ratio = 17, p<0.05). The most informative 4 probes were TP63, MYC, CEP3 and CEP6. The combination of these 4 probes offered a sensitivity of 82% for lung cancer and a specificity of 58%. These results indicate that specific cytogenetic alterations present in preinvasive lung lesions are closely associated with the diagnosis of lung cancer and may therefore have value in assessing lung cancer risk.     

The Fibrin Matrix Regulates Angiogenic Responses within the Hemostatic Microenvironment through Biochemical Control

Conceptually, premature initiation of post-wound angiogenesis could interfere with hemostasis, as it relies on fibrinolysis. The mechanisms facilitating orchestration of these events remain poorly understood, however, likely due to limitations in discerning the individual contribution of cells and extracellular matrix. Here, we designed an in vitro Hemostatic-Components-Model (HCM) to investigate the role of the fibrin matrix as protein factor-carrier, independent of its cell-scaffold function. After characterizing the proteomic profile of HCM-harvested matrix releasates, we demonstrate that the key pro-/anti-angiogenic factors, VEGF and PF4, are differentially bound by the matrix. Changing matrix fibrin mass consequently alters the balance of releasate factor concentrations, with differential effects on basic endothelial cell (EC) behaviors. While increasing mass, and releasate VEGF levels, promoted EC chemotactic migration, it progressively inhibited tube formation, a response that was dependent on PF4. These results indicate that the clot’s matrix component initially serves as biochemical anti-angiogenic barrier, suggesting that post-hemostatic angiogenesis follows fibrinolysis-mediated angiogenic disinhibition. Beyond their significance towards understanding the spatiotemporal regulation of wound healing, our findings could inform the study of other pathophysiological processes in which coagulation and angiogenesis are prominent features, such as cardiovascular and malignant disease.     

Interleukin-6 and alpha-2-macroglobulin indicate an acute-phase state in Alzheimer's disease cortices.

Recent studies indicated that the formation of a major constituent of Alzheimer's disease (AD) senile plaques, called beta A4-peptide, does not result from normal processing of its precursor, amyloid precursor protein (APP). Since proteolytic cleavage of APP inside its beta A4 sequence was found to be part of APP processing the formation of the beta A4-peptide seems to be prevented under normal conditions. We considered whether in AD one of the endogenous proteinase inhibitors might interfere with APP processing. After we had recently found that cultured human neuronal cells synthesize the most potent of the known human proteinase inhibitors, alpha-2-macroglobulin (alpha 2M), upon stimulation with the inflammatory mediator interleukin-6 (IL-6) we now investigated whether alpha 2M and IL-6 could be detected in AD brains. Here we report that AD cortical senile plaques display strong alpha 2M and IL-6 immunoreactivity while no such immunoreactivity was found in age-matched control brains. Strong perinuclear alpha 2M immunoreactivity in hippocampal CA1 neurons of Alzheimer's disease brains indicates that neuronal cells are the site of alpha 2M synthesis in AD brains. We did not detect elevated IL-6 or alpha 2M levels in the cerebrospinal fluid of AD patients. Our data indicate that a sequence of immunological events which seem to be restricted to the local cortical environment is part of AD pathology.     

A low-spin iron complex in human melanoma and rat hepatoma cells and a high-spin iron(II) complex in rat hepatoma cells.

Human melanoma and rat hepatoma cells cultured in the presence of low concentrations (2.5 microM) of low-molecular-weight iron (Fe) chelates and Fe-transferrin complexes have been studied with 57Fe M?ssbauer spectroscopy. The spectra show that holoferritin is only a minor fraction of the total iron present in the cells. The major form of Fe was in a low-spin state unlike the high-spin Fe(III) found in ferritin. Only about 10% of the Fe could be attributed to ferritin. In addition, the hepatoma cells had a high-spin Fe(II) spectral component which made up about 20% of the Fe present.     

Identification of functional PDZ domain binding sites in several human proteins

TIP-15 was previously identified as a cellular protein that can bind to the C-terminal end of the HTLV-1 Tax protein via its two PDZ domains. The sequence of the N-terminal part of TIP-15 is identical to that of the synaptic protein PSD-95. Both proteins are likely to be produced from the same gene by alternative splicing. Whereas expression of the PSD-95 mRNA was detected only with brain RNAs, that of TIP-15 was detected with RNAs from thymus, brain, skeletal muscle and Jurkat cells. The TIP-15 protein exhibits an apparent molecular weight of 40 kD and is weakly expressed in T cell lines. A two-hybrid screen performed with TIP-15 as bait revealed the presence of a PDZ binding site (PDZ-BS) in the following proteins: Lysyl tRNA synthetase, 6-phosphogluconolactonase (6-GPL), Stress-activated protein kinase 3 (SAPK3), NET-1, Diacylglycerol kinase zeta, MTMR1, MCM7, and hSec8. The sequence at the C-terminal ends of these proteins matches the X-S/T-X-V-COOH consensus previously defined for PDZ-BSs, with the exception of 6-GPL and SAPK3 which include a leucine as the C-terminal residue. For Lysyl tRNA synthetase, NET1, MTMR1 and hSec8, binding to TIP-15 was confirmed by co-immunoprecipitation experiments performed with the extracts of transfected COS7 cells. These results show the existence of functional PDZ-BSs in these proteins, but future studies will be necessary to establish whether or not TIP-15 represents a physiological partner. The significance of the presence of a PDZ-BS in these various proteins is discussed with respect to their function.     

Geometric morphometrics applied to viscerocranial bones in three populations of the Lake Tanganyika cichlid fish Tropheus moorii

Lake Tanganyika comprises the oldest and most diverse species flock of cichlid fishes. Many species are subdivided into numerous populations, often classified as geographical races, colour morphs or sister species. Unlike younger species flocks, in which speciation is accompanied by eco-morphological diversification that of Lake Tanganyika is at a mature stage with little further morphological change, most probably caused by stabilizing selection. This study addresses body shape differences among three genetically distinct but morphologically similar populations of Tropheus  moorii , by focusing on bony structures of the cichlid head. We test by means of geometric morphological methods whether shape changes in the cichlid head are based on particular osteological differences. Specimens were disarticulated enzymatically, and standardized digital images of the disarticulated bony elements were taken. A landmark system was established for the dentary, articular, premaxilla, quadrate and the preopercle. Only the dentary shows significant differentiation among the three populations. Since all three populations live in similar cobble habitats and occupy the same trophic niche, the observed difference in the shape of the dentary can either be explained by different directional selection due to subtle habitat differences, or by neutral drift constrained by borders enforced through stabilizing selection. Lack of difference might indicate stabilizing selection on bone shape.