Preparative scale purification of fucosyl-<Emphasis Type="Italic">N</Emphasis>-acetylglucosamine disaccharides and their evaluation as potential prebiotics and antiadhesins

Fucosyl-N-acetylglucosamine disaccharides are important core structures that form part of human mucosal and milk glyco-complexes. We have previously shown that AlfB and AlfC α-L-fucosidases from Lactobacillus casei are able to synthesize fucosyl-α-1,3--N-acetylglucosamine (Fuc-α1,3-GlcNAc) and fucosyl-α-1,6-N-acetylglucosamine (Fuc-α1,6-GlcNAc), respectively, in transglycosylation reactions. Here, these reactions were performed in a semipreparative scale, and the produced disaccharides were purified. The maximum yields obtained of Fuc-α1,3-GlcNAc and Fuc-α1,6-GlcNAc were 4.2 and 9.3 g/l, respectively. The purified fucosyl-disaccharides were then analyzed for their prebiotic effect in vitro using strains from the Lactobacillus casei/paracasei/rhamnosus group and from Bifidobacterium species. The results revealed that 6 out of 11?L. casei strains and 2 out of 6?L. rhamnosus strains tested were able to ferment Fuc-α1,3-GlcNAc, and L. casei BL87 and L. rhamnosus BL327 strains were also able to ferment Fuc-α1,6-GlcNAc. DNA hybridization experiments suggested that the metabolism of Fuc-α1,3-GlcNAc in those strains relies in an α-L-fucosidase homologous to AlfB. Bifidobacterium breve and Bibidobacterium pseudocatenolatum species also metabolized Fuc-α1,3-GlcNAc. Notably, L-fucose was excreted from all the Lactobacillus and Bifidobacterium strains fermenting fucosyl-disaccharides, except from strains L. rhamnosus BL358 and BL377, indicating that in these latest strains, L-fucose was catabolized. The fucosyl-disaccharides were also tested for their inhibitory potential of pathogen adhesion to human colon adenocarcinoma epithelial (HT29) cell line. Enteropathogenic Escherichia coli (EPEC) strains isolated from infantile gastroenteritis were used, and the results showed that both fucosyl-disaccharides inhibited adhesion to different extents of certain EPEC strains to HT29 cells in tissue culture.     

Metabolic adaptation of Pseudomonas pseudoalcaligenes CECT5344 to cyanide: role of malate-quinone oxidoreductases, aconitase and fumarase isoenzymes

In general, the biodegradation of a toxic compound by a micro-organism requires the concurrence of, at least, two features in the biological system: first, the capability of the micro-organism to metabolize the toxic compound, and secondly, the capacity to resist its toxic effect. Pseudomonas pseudoalcaligenes CECT5344 is a bacterium used in the biodegradation of cyanide because it is capable to use it as a nitrogen source. The present review is mainly focused on the putative role of iron-containing enzymes of the tricarboxylic acid cycle in cyanide resistance by P. pseudoalcaligenes CECT5344.     

Stem cells in brain tumorigenesis and their impact on therapy

The concept that the genesis of new cells in the adult mammalian brain is negligible has long influenced our perception and understanding of the origin and development of central nervous system (CNS) tumors. The discovery that neurons and glia are produced throughout life from neural stem cells provides new possibilities for candidate precursor cells of CNS neoplasms. The emerging hypothesis is that alterations in the cellular and genetic mechanisms that control adult neurogenesis might contribute to brain tumorigenesis. As such, opportunities become available to identify new therapeutic strategies.     

Robust reproducible resting state networks in the awake rodent brain

Resting state networks (RSNs) have been studied extensively with functional MRI in humans in health and disease to reflect brain function in the un-stimulated state as well as reveal how the brain is altered with disease. Rodent models of disease have been used comprehensively to understand the biology of the disease as well as in the development of new therapies. RSN reported studies in rodents, however, are few, and most studies are performed with anesthetized rodents that might alter networks and differ from their non-anesthetized state. Acquiring RSN data in the awake rodent avoids the issues of anesthesia effects on brain function. Using high field fMRI we determined RSNs in awake rats using an independent component analysis (ICA) approach, however, ICA analysis can produce a large number of components, some with biological relevance (networks). We further have applied a novel method to determine networks that are robust and reproducible among all the components found with ICA. This analysis indicates that 7 networks are robust and reproducible in the rat and their putative role is discussed.     

Breaking Caenorhabditis elegans the easy way using the Balch homogenizer: An old tool for a new application

The nematode Caenorhabditis elegans is a model organism best known for its powerful genetics. There is an increasing need in the worm community to couple genetics with biochemistry. Isolation of functionally active proteins or nucleic acids without the use of strong oxidizing denaturants or of subcellular compartments from C. elegans has, however, been challenging because of the worms’ thick surrounding cuticle. The Balch homogenizer is a tool that has found much use in mammalian cell culture biology. The interchangeable single ball-bearing design of this instrument permits rapid permeabilization, or homogenization, of cells. Here we demonstrate the utility of the Balch homogenizer for studies with C. elegans. We describe procedures for the efficient breakage and homogenization of every larval stage, including dauers, and show that the Balch homogenizer can be used to extract functionally active proteins. Enzymatic assays for catalase and dihydrolipoamide dehydrogenase show that sample preparation using the Balch homogenizer equals or outperforms conventional methods employing boiling, sonication, or Dounce homogenization. We also describe phenol-free techniques for isolation of genomic DNA and RNA. Finally, we used the tool to isolate coupled mitochondria and polysomes. The reusable Balch homogenizer represents a quick and convenient solution for undertaking biochemical studies on C. elegans.     

An approach to localizing corneal pain representation in human primary somatosensory cortex

The cornea has been a focus of animal electrophysiological research for decades, but little is known regarding its cortical representation in the human brain. This study attempts to localize the somatotopic representation of the cornea to painful stimuli in human primary somatosensory cortex using functional magnetic resonance imaging (fMRI). In this case study, a subject was imaged at 3T while bright light was presented in a block-design, which either produced pain and blinking (during photophobia) or blinking alone (after recovery from photophobia). Pain and blinking produced precisely localized activations in primary somatosensory cortex and primary motor cortex. These results indicate that noxious stimulation of the cornea can produce somatotopic activation in primary somatosensory cortex. This finding opens future avenues of research to evaluate the relationship between corneal pain and central brain mechanisms relating to the development of chronic pain conditions, such as dry eye-like symptoms.     

Yohimbine-induced amygdala activation in pathological gamblers: a pilot study

Rationale and Objectives

There is evidence that drug addiction is associated with increased physiological and psychological responses to stress. In this pilot functional magnetic resonance imaging (fMRI) study we assessed whether a prototype behavioral addiction, pathological gambling (PG), is likewise associated with an enhanced response to stress.

Methods

We induced stress by injecting yohimbine (0.2–0.3 mg/kg, IV), an alpha-2 adrenoceptor antagonist that elicits stress-like physiological and psychological effects in humans and in laboratory animals, to four subjects with PG and to five non-gamblers mentally healthy control subjects. Their fMRI brain responses were assessed along with subjective stress and gambling urges ratings.

Results

Voxelwise analyses of data sets from individual subjects, utilizing generalized linear model approach, revealed significant left amygdala activation in response to yohimbine across all PG subjects. This amygdala effect was not observed in the five control individuals. Yohimbine elicited subjective stress ratings in both groups with greater (albeit not statically significantly) average response in the PG subjects. On the other hand, yohimbine did not induce urges to gamble.

Conclusions

The present data support the hypothesis of brain sensitization to pharmacologically-induced stress in PG.     

Cardiac and vascular phenotypes in the apolipoprotein E-deficient mouse

Cardiovascular death is frequently associated with atherosclerosis, a chronic multifactorial disease and a leading cause of death worldwide. Genetically engineered mouse models have proven useful for the study of the mechanisms underlying cardiovascular diseases. The apolipoprotein E-deficient mouse has been the most widely used animal model of atherosclerosis because it rapidly develops severe hypercholesterolemia and spontaneous atherosclerotic lesions similar to those observed in humans. In this review, we provide an overview of the cardiac and vascular phenotypes and discuss the interplay among nitric oxide, reactive oxygen species, aging and diet in the impairment of cardiovascular function in this mouse model.     

Challenges and Solutions in the Development of Genomic Biomarker Panels: A Systematic Phased Approach

In the post-genome era, high throughput gene expression profiling has been successfully used to develop genomic biomarker panels (GBP) that can be integrated into clinical decision making. The development of GBPs in the context of personalized medicine is a scientifically challenging and resource-intense process. It needs to be accomplished in a systematic phased approach to address biological variation related to a clinical phenotype (e.g. disease etiology, gender, etc.) and minimize technical variation (noise). Here we present the methodological aspects of GBP development based on the experience of the Cardiac Allograft Rejection Gene Expression Observation (CARGO) study, a study that lead to the development of a molecular classifier for rejection screening in heart transplant patients.     

Emergence of a small-world functional network in cultured neurons

The functional networks of cultured neurons exhibit complex network properties similar to those found in vivo. Starting from random seeding, cultures undergo significant reorganization during the initial period in vitro, yet despite providing an ideal platform for observing developmental changes in neuronal connectivity, little is known about how a complex functional network evolves from isolated neurons. In the present study, evolution of functional connectivity was estimated from correlations of spontaneous activity. Network properties were quantified using complex measures from graph theory and used to compare cultures at different stages of development during the first 5 weeks in vitro. Networks obtained from young cultures (14 days in vitro) exhibited a random topology, which evolved to a small-world topology during maturation. The topology change was accompanied by an increased presence of highly connected areas (hubs) and network efficiency increased with age. The small-world topology balances integration of network areas with segregation of specialized processing units. The emergence of such network structure in cultured neurons, despite a lack of external input, points to complex intrinsic biological mechanisms. Moreover, the functional network of cultures at mature ages is efficient and highly suited to complex processing tasks.