Expression of vesicle-associated membrane-protein-associated protein B cleavage products in peripheral blood leukocytes and cerebrospinal fluid of patients with sporadic amyotrophic lateral sclerosis.

BACKGROUND AND PURPOSE: Vesicle-associated membrane-protein-associated protein B (VAPB) is an endoplasmic reticulum (ER) resident protein participating in ER function, vesicle trafficking, calcium homeostasis and lipid transport. Its N-terminal domain, named MSP, is cleaved and secreted, serving as an extracellular ligand. VAPB mutations are linked to autosomal-dominant motor neuron diseases, including amyotrophic lateral sclerosis (ALS) type 8. An altered VAPB function is also suspected in sporadic ALS (SALS). METHODS: The expression pattern of VAPB cleavage and secreted products in the peripheral blood leukocytes (PBL) and cerebrospinal fluid (CSF) of SALS patients and neurological controls was assessed. PBL from healthy controls were also analyzed. Assays were carried out through western blotting, using an anti-VAPB (N-terminal) antibody. RESULTS: Two VAPB fragments containing the MSP domain (17 kDa and 14 kDa molecular sizes) were identified in PBL of SALS and controls, with no significant differences amongst groups. In CSF, only the 14 kDa VAPB MSP fragment was expressed and a corresponding VAPA fragment was not detected. The CSF VAPB fragment was absent in 58.7% of SALS patients, of whom 79.2% were bulbar onset (P = 0.001, bulbar versus spinal). CONCLUSIONS: The absence of the CSF VAPB MSP fragment from most bulbar-onset SALS patients suggests a specific alteration of brain-derived VAPB cleavage and secretion in this group of patients, and hints at a role of VAPB in the pathophysiology of this motor neuron disease.

Heat-Resistant Aphanizomenon flos-aquae (AFA) Extract (Klamin®) as a Functional Ingredient in Food Strategy for Prevention of Oxidative Stress.

Microalgae are generally considered an excellent source of vitamins, minerals, and bioactive molecules that make them suitable to be introduced in cosmetics, pharmaceuticals, and food industries. Aphanizomenon flos-aquae (AFA), an edible microalga, contains numerous biomolecules potentially able to prevent some pathologies including age-related disorders. With the aim to include an AFA extract (Klamin®) as a functional ingredient in baked products, we investigated if its bioactive molecules are destroyed or inactivated after standard cooking temperature. The AFA extract was exposed to heat stress (AFA-HS), and no significant decrease in pigment, polyphenol, and carotenoid content was detected by spectroscopic analysis. Thermal stability of AFA-HS extract was demonstrated by thermogravimetric analysis (TGA), and no change in the morphology of the granules of the powder was noticed by SEM microscopic observation. By Folin-Ciocalteu, ORAC, and ABTS assays, no change in the antioxidant activity and polyphenol contents was found after high-temperature exposition. When added in cell culture, solubilized AFA-HS lost neither its scavenging ability against ROS generation nor its protective role against Abeta, the main peptide involved in Alzheimer's disease. Prebiotic and antioxidant activities of AFA extract that are not lost after thermal stress were verified on E. coli bacteria. Finally, AFA-HS cookies, containing the extract as one of their ingredients, showed increased polyphenols. Here, we evaluate the possibility to use the AFA extract to produce functional food and prevent metabolic and age-related diseases.

Effects of the Aphanizomenon flos-aquae Extract (Klamin®) on a Neurodegeneration Cellular Model.

Cyanobacteria have been recognized as a source of bioactive molecules to be employed in nutraceuticals, pharmaceuticals, and functional foods. An extract of Aphanizomenon flos-aquae (AFA), commercialized as Klamin®, was subjected to chemical analysis to determine its compounds. The AFA extract Klamin® resulted to be nontoxic, also at high doses, when administered onto LAN5 neuronal cells. Its scavenging properties against ROS generation were evaluated by using DCFH-DA assay, and its mitochondrial protective role was determined by JC-1 and MitoSOX assays. Klamin® exerts a protective role against beta amyloid- (Aß-) induced toxicity and against oxidative stress. Anti-inflammatory properties were demonstrated by NFßB nuclear localization and activation of IL-6 and IL-1ß inflammatory cytokines through ELISA. Finally, by using thioflavin T (ThT) and fluorimetric measures, we found that Klamin® interferes with Aß aggregation kinetics, supporting the formation of smaller and nontoxic structures compared to toxic Aß aggregates alone. Altogether, these data indicate that the AFA extract may play a protective role against mechanisms leading to neurodegeneration.

Neurosteroids in the retina: neurodegenerative and neuroprotective agents in retinal degeneration.

Steroids may have a powerful role in neuronal degeneration. Recent research has revealed that steroids may influence the onset and progression of some retinal disorders as well as neurodegenerative diseases and, as in brain, they accumulate in the retina via a local synthesis (neurosteroids) and metabolism of blood-circulating steroid hormones. Their crucial role as neurodegenerative and neuroprotective agents has been also upheld in a retinal excitotoxic paradigm. These findings are reviewed especially from the emerging perspective that after an insult local changes in steroidogenic responses and consequent neurosteroid availability might turn out to be offensive or defensive cellular adaptations for the potentiation or prevention of neuronal death.

17beta-estradiol synthesis in the adult male rat retina.

17beta-Estradiol (E2) exerts neurotrophic and neuroprotective effects in the retina as well as in other CNS structures, independently of sex. Retinal effects, however, have not been supported by evidence on local synthesis, and whether CNS 17beta-estradiol is formed in a neurosteroidogenic pathway starting from cholesterol conversion into pregnenolone is a question still left unanswered. In the adult male rat retina, we have previously showed localization and activity of the P450 side chain cleavage (P450scc) enzyme, which is involved in pregnenolone synthesis. Here, we demonstrate both the mRNA and protein expression of 3beta-hydroxysteroid dehydrogenase (3beta-HSD), P450aromatase and also of P450scc, but only the protein expression of P450 17alpha-hydroxylase/lyase (P450c17). Using radiolabeled pregnenolone and testosterone as precursors, in the isolated and intact retina of adult male rats, E2 is produced in a large amount by each precursor within 1-4h, suggesting a highly active metabolic pathway towards its formation. The immunolocalization pattern shows enzymes and estrogen receptor subtypes (ERalpha, ERbeta) scattered in the retina with different intensities throughout the layers. The results point to the adult male rat retina as a neurosteroidogenic structure where E2 synthesis via a progesterone pathway and the presence of estrogen receptors provide important clues for understanding the neurotrophic and neuroprotective effects of the steroid hormone.

Digital processing of dual-plate speckle photography data.

Dual-plate speckle photography provides a technique for canceling rigid body displacements of an object by recording the images that correspond to the two deformation states on separate photographic plates. When both plates are positioned with a glass base between the emulsions and illuminated by a laser beam, a pattern of circular fringes is formed. Speckle displacement can be determined by measuring the distance between the fringe pattern center and the undiffracted laser beam. The performance of an algorithm for automatic analysis of these fringes is evaluated by using computer-generated fringe patterns. Accuracy is determined for different values of speckle displacements and fringe visibilities.

Unwrapping of interferometric phase-fringe maps by the discrete cosine transform.

Current whole-field interferometric techniques yield a phase distribution in modulo 2π. Removal of the resulting cyclic discontinuities is a process known as unwrapping, which must be performed before the data can be interpreted. We investigate an iterative unwrapping technique recently published by Ghiglia and Romero [J. Opt. Soc. Am. A 11, 107 (1994)], which is based on least-squares minimization, obtained by the discrete cosine transform. We apply this technique to remove phase wraps from electronic speckle pattern interferometry data, using modest personal computer hardware. The algorithm is shown to be fast, easy to implement, robust in the presence of noise, and able to handle phase inconsistencies without propagating local errors.

Evaluation of a preconditioned conjugate-gradient algorithm for weighted least-squares unwrapping of digital speckle-pattern interferometry phase maps.

Inasmuch as current fringe analysis techniques used in digital speckle-pattern interferometry (DSPI) yield a phase map modulo 2pi, phase unwrapping is the final step of any data evaluation process. The performance of a recently published algorithm used to unwrap DSPI phase maps is investigated. The algorithm is based on a least-squares minimization technique that is solvable by the discrete cosine transform. When phase inconsistencies are present, they are handled by exclusion of invalid pixels from the unwrapping process through the assignment of zero-valued weights. Then the weighted unwrapping problem is solved in an iterative manner by a preconditioned conjugate-gradient method. The evaluation is carried out with computer-simulated DSPI phase maps, an approach that permits the generation of phase fields without inconsistencies, which are then used to calculate phase deviations as a function of the iteration number. Real data are also used to illustrate the performance of the algorithm.

Unwrapping of Digital Speckle-Pattern Interferometry Phase Maps by use of a Minimum L(0)-Norm Algorithm.

The performance of a minimum L(0)-norm unwrapping algorithm is investigated by use of synthetic digital speckle-pattern interferometry (DSPI) wrapped phase maps that simulate experimentally obtained data. This algorithm estimates its own weights to mask inconsistent pixels. Particular features usually included in DSPI wrapped phase distributions, such as shears, speckle noise, fringe cuts, object physical limits, and superimposed phase maps, are analyzed. Some adequate approaches to solving these features are discussed. Finally, it is shown that a complex case in which shears and fringe cuts coexist in the wrapped phase cannot be solved satisfactorily with the minimum L(0)-norm algorithm by itself. To cope with this problem, we propose a new scheme.

Holographic investigation of residual deformations induced by a pulsed ion implanter.

A new use of holographic interferometry to investigate the residual deformations induced in nitrogen implanted specimens by a plasma focus device is reported. The method is simple and nondestructive. Experimental results obtained for AISI 304 stainless steel specimens are presented.