Maintenance of syntenic groups between Cathartidae and Gallus gallus indicates symplesiomorphic karyotypes in new world vultures

Similarities between New World and Old World vultures have been interpreted to reflect a close relationship and to suggest the inclusion of both in Accipitridae (Falconiformes). However, deeper analyses indicated that the placement of the New World vultures (cathartids) in this Order is uncertain. Chromosome analysis has shown that cathartids retained a karyotype similar to the putative avian ancestor. In order to verify the occurrence of intrachromosomal rearrangements in cathartids, we hybridized whole chromosome probes of two species (Gallus gallus and Leucopternis albicollis) onto metaphases of Cathartes aura. The results showed that not only were the syntenic groups conserved between Gallus and C. aura, but probably also the general gene order, suggesting that New World vultures share chromosomal symplesiomorphies with most bird lineages.     

Mapping phosphoproteins in Neisseria meningitidis serogroup A

To investigate the phosphorylation capability of serogroup A Neisseria meningitidis (MenA) and to implement our knowledge in meningococcal biology and in bacterial post-translational modifications, cell extracts were separated by 2-DE and 51 novel phosphoproteins were revealed by the use of the highly specific Ser/Thr/Tyr-phosphorylated proteins staining by Pro-Q Diamond and identified by MALDI-ToF/MS. Our results indicate that phosphorylation in MenA is comparable to that of other bacterial species. A first functional characterization of the identified modified proteins was also given, in order to understand their role in meningococcal physiopathology.     

In vivo Near Infrared Fluorescence (NIRF) Intravascular Molecular Imaging of Inflammatory Plaque,a Multimodal Approach to Imaging of Atherosclerosis

The vascular response to injury is a well-orchestrated inflammatory response triggered by the accumulation of macrophages within the vessel wall leading to an accumulation of lipid-laden intra-luminal plaque, smooth muscle cell proliferation and progressive narrowing of the vessel lumen. The formation of such vulnerable plaques prone to rupture underlies the majority of cases of acute myocardial infarction. The complex molecular and cellular inflammatory cascade is orchestrated by the recruitment of T lymphocytes and macrophages and their paracrine effects on endothelial and smooth muscle cells.¹Molecular imaging in atherosclerosis has evolved into an important clinical and research tool that allows in vivo visualization of inflammation and other biological processes. Several recent examples demonstrate the ability to detect high-risk plaques in patients, and assess the effects of pharmacotherapeutics in atherosclerosis.⁴ While a number of molecular imaging approaches (in particular MRI and PET) can image biological aspects of large vessels such as the carotid arteries, scant options exist for imaging of coronary arteries.² The advent of high-resolution optical imaging strategies, in particular near-infrared fluorescence (NIRF), coupled with activatable fluorescent probes, have enhanced sensitivity and led to the development of new intravascular strategies to improve biological imaging of human coronary atherosclerosis.Near infrared fluorescence (NIRF) molecular imaging utilizes excitation light with a defined band width (650-900 nm) as a source of photons that, when delivered to an optical contrast agent or fluorescent probe, emits fluorescence in the NIR window that can be detected using an appropriate emission filter and a high sensitivity charge-coupled camera. As opposed to visible light, NIR light penetrates deeply into tissue, is markedly less attenuated by endogenous photon absorbers such as hemoglobin, lipid and water, and enables high target-to-background ratios due to reduced autofluorescence in the NIR window. Imaging within the NIR ''window'' can substantially improve the potential for in vivo imaging.^2,5Inflammatory cysteine proteases have been well studied using activatable NIRF probes¹⁰, and play important roles in atherogenesis. Via degradation of the extracellular matrix, cysteine proteases contribute importantly to the progression and complications of atherosclerosis⁸. In particular, the cysteine protease, cathepsin B, is highly expressed and colocalizes with macrophages in experimental murine, rabbit, and human atheromata.^3,6,7 In addition, cathepsin B activity in plaques can be sensed in vivo utilizing a previously described 1-D intravascular near-infrared fluorescence technology⁶, in conjunction with an injectable nanosensor agent that consists of a poly-lysine polymer backbone derivatized with multiple NIR fluorochromes (VM110/Prosense750, ex/em 750/780nm, VisEn Medical, Woburn, MA) that results in strong intramolecular quenching at baseline.¹⁰ Following targeted enzymatic cleavage by cysteine proteases such as cathepsin B (known to colocalize with plaque macrophages), the fluorochromes separate, resulting in substantial amplification of the NIRF signal. Intravascular detection of NIR fluorescence signal by the utilized novel 2D intravascular NIRF catheter now enables high-resolution, geometrically accurate in vivo detection of cathepsin B activity in inflamed plaque. In vivo molecular imaging of atherosclerosis using catheter-based 2D NIRF imaging, as opposed to a prior 1-D spectroscopic approach,⁶ is a novel and promising tool that utilizes augmented protease activity in macrophage-rich plaque to detect vascular inflammation.^11,12 The following research protocol describes the use of an intravascular 2-dimensional NIRF catheter to image and characterize plaque structure utilizing key aspects of plaque biology. It is a translatable platform that when integrated with existing clinical imaging technologies including angiography and intravascular ultrasound (IVUS), offers a unique and novel integrated multimodal molecular imaging technique that distinguishes inflammatory atheromata, and allows detection of intravascular NIRF signals in human-sized coronary arteries.Download video file.^{(61M, mov)}     

Different factors affecting human ANP amyloid aggregation and their implications in congestive heart failure

Aims

Atrial Natriuretic Peptide (ANP)-containing amyloid is frequently found in the elderly heart. No data exist regarding ANP aggregation process and its link to pathologies. Our aims were: i) to experimentally prove the presumptive association of Congestive Heart Failure (CHF) and Isolated Atrial Amyloidosis (IAA); ii) to characterize ANP aggregation, thereby elucidating IAA implication in the CHF pathogenesis.

Methods and Results

A significant prevalence (85%) of IAA was immunohistochemically proven ex vivo in biopsies from CHF patients. We investigated in vitro (using Congo Red, Thioflavin T, SDS-PAGE, transmission electron microscopy, infrared spectroscopy) ANP fibrillogenesis, starting from α-ANP as well as the ability of dimeric β-ANP to promote amyloid formation. Different conditions were adopted, including those reproducing β-ANP prevalence in CHF. Our results defined the uncommon rapidity of α-ANP self-assembly at acidic pH supporting the hypothesis that such aggregates constitute the onset of a fibrillization process subsequently proceeding at physiological pH. Interestingly, CHF-like conditions induced the production of the most stable and time-resistant ANP fibrils suggesting that CHF affected people may be prone to develop IAA.

Conclusions

We established a link between IAA and CHF by ex vivo examination and assessed that β-ANP is, in vitro, the seed of ANP fibrils. Our results indicate that β-ANP plays a crucial role in ANP amyloid deposition under physiopathological CHF conditions. Overall, our findings indicate that early IAA-related ANP deposition may occur in CHF and suggest that these latter patients should be monitored for the development of cardiac amyloidosis.     

Evidence of involvement of leptin and IL-6 peptides in the action of interferon-beta in secondary progressive multiple sclerosis

Leptin is a peptide hormone which acts on cells of immune system by influencing the production of cytokines. Serum leptin levels and cytokine production by peripheral blood mononuclear cells (PBMC) were measured in 18 secondary progressive multiple sclerosis (SPMS) patients under IFN-beta-1b treatment. There were no overall effects on leptin, interleukin-6 (IL-6), IL-10 and IL-12 p40 after 2, 6 and 12 months of treatment. However, leptin and IL-6 decreased after 6 and 12 months of treatment in 12 patients who did not show progression of disability. Thus, our pilot data show that the beneficial effect of IFN-beta on some SPMS patients might be associated with the reduced levels of leptin and reduced IL-6 production by PBMC.     

Antisense reduction of thylakoidal ascorbate peroxidase in <Emphasis Type="Italic">Arabidopsis</Emphasis> enhances Paraquat-induced photooxidative stress and Nitric Oxide-induced cell death

The production and characterization of Arabidopsis plants containing a transgene in which the Arabidopsis tAPX is inserted in antisense orientation, is described. tAPX activity in these transgenic tAPX plants is around 50% of control level. The tAPX antisense plants are phenotypically indistinguishable from control plants under normal growth conditions; they show, however, enhanced sensitivity to the O₂^–-generating herbicide, Paraquat. Interestingly, the tAPX antisense plants show enhanced symptoms of damage when cell death is triggered through treatment with the nitric oxide-donor, SNP. These results are in accordance with the ones recently obtained with transgenic plants overexpressing tAPX; altogether, they suggest that tAPX, besides the known ROS scavenging role, is also involved in the fine changes of H₂O₂ concentration during signaling events.     

Monitoring of resistance to the pyrethroid cypermethrin in Brazilian Aedes aegypti (Diptera: Culicidae) populations collected between 2001 and 2003

Resistance to cypermethrin of different Aedes aegypti Brazilian populations, collected at two successive periods (2001 and 2002/2003), was monitored using the insecticide-coated bottles bioassay. Slight modifications were included in the method to discriminate between mortality and the knock down effect. Although this pyrethroid was recently started to be used in the country to control the dengue vector, a decrease in susceptibility was noted between both periods analyzed, particularly in the city of Rio de Janeiro. The results indicate that resistance is due at least in part to a target site alteration.     

Glycomimetics as decorating motifs for oligonucleotides: solid-phase synthesis, stability, and hybridization properties of carbopeptoid-oligonucleotide conjugates

The online solid-phase synthesis of oligonucleotides conjugated at the 3' end with [1-6]-linked oligosaccharide mimics having the O-glycosidic linkages replaced by amide bonds is here described. The assembly of the carbohydrate domain has been carried out by exploiting classical solid phase peptide synthetic protocols, starting from solid supports functionalized with 1-azido sugars, in association with suitably protected 1-azido uronic acids of glucose and lactose, chosen as model addition monomers. After the insertion of a flexible linker, elongation of the oligodeoxyribonucleotide (ODN) chain was performed by standard automated phosphoramidite protocols. 3'-Glycoconjugated 18-mers exhibited an increased enzymatic stability with respect to the same unmodified ODN sequence. UV thermal denaturation experiments showed that the presence of the oligosaccharide tail at the 3' end of the oligonucleotides did not negatively interfere with their duplex formation abilities.     

A new polymeric coating for protein microarrays

Despite the increasing interest in arraying proteins in a high-density format, several technical issues still impede the development of protein microarray technology. One of the major problems is the availability of substrates that are able to bind native proteins with high density. In this study, we investigated the suitability of a novel surface as a support for protein microarrays. A polymeric glass coating is obtained by physical adsorption of a N,N-dimethylacrylamide (DMA), N,N-acryloyloxysuccinimide (NAS), and [3-(methacryloyl-oxy)propyl]trimethoxysilyl (MAPS) copolymer. The coating procedure provides a fast and inexpensive method of producing hydrophilic functional surfaces. The slide performance was investigated in a protein-protein interaction experiment and in the assessment of rheumatoid factor (RF) in human serum samples. The results demonstrate that the ligands immobilized on the polymeric surface maintain an active conformation and are easily accessible, providing a detection limit of 54amol/spot. Moreover, in the RF assay, after hybridization with the sera, the slides have a low background, leading to a detection limit of 900amol/spot.