Domenico Garozzo

β-Amyloid Monomers Are Neuroprotective

The 42-aa-long β-amyloid protein—Aβ1-42—is thought to play a central role in the pathogenesis of Alzheimers disease (AD) (Walsh and Selkoe, 2007). Data from AD brain (Shankar et al., 2008), transgenic APP (amyloid precursor protein)-overexpressing mice (Lesné et al., 2006), and neuronal cultures treated with synthetic Aβ peptides (Lambert et al., 1998) indicate that self-association of Aβ1-42 monomers into soluble oligomers is required for neurotoxicity. The function of monomeric Aβ1-42 is unknown. The evidence that Aβ1-42 is present in the brain and CSF of normal individuals suggests that the peptide is physiologically active (Shoji, 2002). Here we show that synthetic Aβ1-42 monomers support the survival of developing neurons under conditions of trophic deprivation and protect mature neurons against excitotoxic death, a process that contributes to the overall neurodegeneration associated with AD. The neuroprotective action of Aβ1-42 monomers was mediated by the activation of the PI-3-K (phosphatidylinositol-3-kinase) pathway, and involved the stimulation of IGF-1 (insulin-like growth factor-1) receptors and/or other receptors of the insulin superfamily. Interestingly, monomers of Aβ1-42 carrying the Arctic mutation (E22G) associated with familiar AD (Nilsberth et al., 2001) were not neuroprotective. We suggest that pathological aggregation of Aβ1-42 may also cause neurodegeneration by depriving neurons of the protective activity of Aβ1-42 monomers. This “loss-of-function” hypothesis of neuronal death should be taken into consideration when designing therapies aimed at reducing Aβ burden.

DPM2-CDG: A muscular dystrophy-dystroglycanopathy syndrome with severe epilepsy

Congenital disorders of glycosylation (CDG) are a group of metabolic diseases due to defects in protein and lipid glycosylation. We searched for the primary defect in 3 children from 2 families with a severe neurological phenotype, including profound developmental delay, intractable epilepsy, progressive microcephaly, severe hypotonia with elevated blood creatine kinase levels, and early fatal outcome. There was clinical evidence of a muscular dystrophy–dystroglycanopathy syndrome, supported by deficient O‐mannosylation by muscle immunohistochemistry.

Primary thermal decomposition processes in aliphatic polyamides

Abstract The primary thermal decomposition processes of a series of aliphatic polyamides were investigated by Direct Chemical Ionization (DCI) and Tandem mass spectrometry. Results show that an intramolecular exchange process is the preferred thermal decomposition mechanism for many of the polyamides. Decomposition through a C-H hydrogen transfer reaction appears to occur as a secondary thermal process, except in the case of nylon 3 because of its particular structure. A quite different decomposition pathway is followed by nylons from adipic acid (nylons 6.6 and 11.6), where specific structural factors make the formation of cyclopentanone preferred. These polymers decompose via a C-H hydrogen transfer to nitrogen with the formation of compounds having amine and/or ketoamide end groups. These primary products further decompose or react with the formation of cyclopentanone and compounds bearing amine and/or Schiff base groups.

Linkage analysis in disaccharides by electrospray mass spectrometry.

Analysis of the structures of complex carbohydrates requires knowledge of the identity, anomeric configuration, and sequence of the sugar residues, and identification of the reducing terminus and the positions of the glycosidic linkages. Desorption-m.s. and f.a.b.-m.s. are powerful techniques for determining the sequence, the pattern of branching, and the molecular weight of oligosaccharides containing up to 30 sugar units, and the structure of the aglycon14. Negative-ion tandem-f.a.b.-m.s. can be used to discriminate between the linkage positions in underivatised oligosaccharides5,6. Electrospray (e.s.) ionisation has also been described7-9. Although most of the applications have been concerned with the determination of molecular weights and sequencing of proteins, some studies have shown that it can be applied to carbohydrates, and we now report its application in the linkage analysis of reducing disaccharides. The negative-ion e-S.-mass spectra of (1~2)(sophorose), (l-+3)(laminaribiose), (1+4)(cellobiose), and (l-+6)(gentiobiose), /I-linked glucodisaccharides shown in Figs. l-4, respectively, reflect the positions of the linkages. Each mass spectrum contains peaks at m/z 34 1 for (M H)and at m/z I79 and 16 1 associated with fragmentation which involves the glycosidic linkages. In addition to these peaks, there are peaks at m/z 323 (sophorose, Fig. l), 28 1 (cellobiose and gentiobiose, Figs. 3 and 4), 263 (sophorose and cellobiose Figs. 1 and 3), 251 (gentiobiose, Fig. 4), and 221 (sophorose, cellobiose, and gentiobiose, Figs. 1,3, and 4). For laminaribiose, only the peaks at m/z 341, 179, and 161 are present (Fig. 2). The peak at m/z 323 is due to loss of water from the (M H)ion, and those at m/z 281, 263, 251, and 221 are associated with fragments from the sugar rings which are diagnostic of the position of the linkage. These fragmentations are likely to involve the reducing moiety, since the non-reducing moieties are identical in the four disaccharides.

Self-Assembly Dynamics of Modular Homoditopic Bis-calix[5]arenes and Long-Chain α,ω-Alkanediyldiammonium Components

Homoditopic building blocks 1, featuring two pi-rich cone-like calix[5]arene moieties connected at their narrow rims by a rigid o-, m-, or p-xylyl spacer in a centrosymmetric divergent arrangement, show a remarkable tendency to spontaneously and reversibly self-assemble with the complementary homoditopic alpha,omega-alkanediyldiammonium dipicrate guest salts C8-C12 x 2Pic through iterative intermolecular inclusion events, forming supramolecular assemblies whose composition and dynamics strongly depend upon the length of the connector, the geometry of the spacer, as well as the concentration and/or molar ratios between the two components. (1)H NMR spectroscopy and ESI-MS studies of 1/C(n) x 2Pic modular homoditopic pairs support the formation of discrete (bis)-endo-cavity assemblies with the shorter C8 and C9 connectors, and/or (poly)capsular assemblies with the longer C10-C12 components under appropriate concentrations and molar ratios (50 mM equimolar solutions). (1)H NMR titration experiments and diffusion NMR studies provide clear evidence for the self-assembly dynamics of the complementary pairs here investigated.

The acylation and phosphorylation pattern of lipid A from Xanthomonas campestris strongly influence its ability to trigger the innate immune response in Arabidopsis.

Lipopolysaccharides (LPSs) are major components of the cell surface of Gram‐negative bacteria. LPSs comprise a hydrophilic heteropolysaccharide (formed by the core oligosaccharide and the O‐specific polysaccharide) that is covalently linked to the glycolipid moiety lipid A, which anchors these macromolecules to the external membrane. LPSs are one of a group of molecules called pathogen‐associated molecular patterns (PAMPs) that are indispensable for bacterial growth and viability, and act to trigger innate defense responses in eukaryotes. We have previously shown that LPS from the plant pathogen Xanthomonas campestris pv. campestris (Xcc) can elicit defense responses in the model plant Arabidopsis thaliana. Here we have extended these studies by analysis of the structure and biological activity of LPS from a nonpathogenic Xcc mutant, strain 8530. We show that this Xcc strain is defective in core completion and introduces significant modification in the lipid A region, which involves the degree of acylation and nonstoichiometric substitution of the phosphate groups with phosphoethanolamine. Lipid A that was isolated from Xcc strain 8530 did not have the ability to induce the defense‐related gene PR1 in Arabidopsis, or to prevent the hypersensitive response (HR) that is caused by avirulent bacteria as the lipid A from the wild‐type could. This suggests that Xcc has the capacity to modify the structure of the lipid A to reduce its activity as a PAMP. We speculate that such effects might occur in wild‐type bacteria that are exposed to stresses such as those that might be encountered during plant colonization and disease.

Covalently linked hopanoid-lipid A improves outer-membrane resistance of a Bradyrhizobium symbiont of legumes

Lipopolysaccharides (LPSs) are major components of the outer membrane of Gram-negative bacteria and are essential for their growth and survival. They act as a structural barrier and play an important role in the interaction with eukaryotic hosts. Here we demonstrate that a photosynthetic Bradyrhizobium strain, symbiont of Aeschynomene legumes, synthesizes a unique LPS bearing a hopanoid covalently attached to lipid A. Biophysical analyses of reconstituted liposomes indicate that this hopanoid-lipid A structure reinforces the stability and rigidity of the outer membrane. In addition, the bacterium produces other hopanoid molecules not linked to LPS. A hopanoid-deficient strain, lacking a squalene hopene cyclase, displays increased sensitivity to stressful conditions and reduced ability to survive intracellularly in the host plant. This unusual combination of hopanoid and LPS molecules may represent an adaptation to optimize bacterial survival in both free-living and symbiotic states.

Multiplexed glycoproteomic analysis of glycosylation disorders by sequential yolk immunoglobulins immunoseparation and MALDI-TOF MS.

This study applied yolk immunoglobulins immunoaffinity separation and MALDI‐TOF MS for clinical proteomics of congenital disorders of glycosylation (CDG) and secondary glycosylation disorders [galactosemia and hereditary fructose intolerance (HFI)]. Serum transferrin (Tf) and α1‐antitrypsin (AAT) that are markers for CDG, were purified sequentially to obtain high‐quality MALDI mass spectra to differentiate single glycoforms of the native intact glycoproteins. The procedure was found feasible for the investigation of protein macroheterogeneity due to glycosylation site underoccupancy then ensuing the characterization of patients with CDG group I (N‐glycan assembly disorders). Following PNGase F digestion of the purified glycoprotein, the characterization of protein microheterogeneity by N‐glycan MS analysis was performed in a patient with CDG group II (processing disorders). CDG‐Ia patients showed a typical profile of underglycosylation where the fully glycosylated glycoforms are always the most abundant present in plasma with lesser amounts of partially and unglycosylated glycoforms in this order. Galactosemia and HFI are potentially fatal diseases, which benefit from early diagnosis and prompt therapeutic intervention. In symptomatic patients with galactosemia and in those with HFI, MALDI MS of Tf and AAT depicts a hypoglycosylation profile with a significant increase of underglycosylated glycoforms that reverses by dietary treatment, representing a clue for diagnosis and treatment monitoring.

Multivalent binding of galactosylated cyclodextrin vesicles to lectin

Amphiphilic beta-cyclodextrins with alkylthio chains at the primary-hydroxyl side and galactosylthio-oligo-(ethylene glycol) units at the secondary-hydroxyl side, which form nanoparticles and vesicles, show multivalent effects in their binding to lectin.

Exopolysaccharides produced by a clinical strain of Burkholderia cepacia isolated from a cystic fibrosis patient

Burkholderia cepacia is an opportunistic pathogen involved in pulmonary infections related to cystic fibrosis. A clinical strain, BTS13, was isolated and the production of exopolysaccharides was tested growing the bacteria on two different media, one of which was rich in mannitol as carbon source. The primary structure of the polysaccharides was determined using mostly mass spectrometry and NMR spectroscopy. On both media an exopolysaccharide having the following repeating unit was produced: -->5)-beta-Kdop-(2-->3)-beta-D-Galp2Ac-(1-->4)-alpha-D-Galp-(1-->3)-beta-D-Galp-(1--> This polysaccharide has already been described as the biosynthetic product of another Burkholderia species, B. pseudomallei, the microbial agent causing melioidosis. In addition to this, when grown on the mannitol-rich medium, B. cepacia strain BTS13 produced another polysaccharide that was established to be levan: -->6)-beta-D-Fruf-(2-->. The content of levan was about 20% (w/w) of the total amount of polymers. The ability of B. cepacia to produce these two exopolysaccharides opens new perspectives in the investigation of the role of polysaccharides in lung infections.