Integrity of White Matter is Compromised in Mice with Hyaluronan Deficiency

Neurochemical Research - Brain white matter is the means of efficient signal propagation in brain and its dysfunction is associated with many neurological disorders. We studied the effect of...     

SNAP-23 regulates phagosome formation and maturation in macrophages

Synaptosomal associated protein of 23 kDa (SNAP-23), a plasma membrane–localized soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE), has been implicated in phagocytosis by macrophages. For elucidation of its precise role in this process, a macrophage line overexpressing monomeric Venus–tagged SNAP-23 was established. These cells showed enhanced Fc receptor–mediated phagocytosis. Detailed analyses of each process of phagocytosis revealed a marked increase in the production of reactive oxygen species within phagosomes. Also, enhanced accumulation of a lysotropic dye, as well as augmented quenching of a pH-sensitive fluorophore were observed. Analyses of isolated phagosomes indicated the critical role of SNAP-23 in the functional recruitment of the NADPH oxidase complex and vacuolar-type H⁺-ATPase to phagosomes. The data from the overexpression experiments were confirmed by SNAP-23 knockdown, which demonstrated a significant delay in phagosome maturation and a reduction in uptake activity. Finally, for analyzing whether phagosomal SNAP-23 entails a structural change in the protein, an intramolecular Förster resonance energy transfer (FRET) probe was constructed, in which the distance within a TagGFP2-TagRFP was altered upon close approximation of the N-termini of its two SNARE motifs. FRET efficiency on phagosomes was markedly enhanced only when VAMP7, a lysosomal SNARE, was coexpressed. Taken together, our results strongly suggest the involvement of SNAP-23 in both phagosome formation and maturation in macrophages, presumably by mediating SNARE-based membrane traffic.     

Development of an HPLC Method with an ODS Column to Determine Low Levels of Aspartame Diastereomers in Aspartame

α-L-Aspartyl-D-phenylalanine methyl ester (L, D-APM) and α-D-aspartyl-L-phenylalanine methyl ester (D, L-APM) are diastereomers of aspartame (N-L-α-Aspartyl-L-phenylalanine-1-methyl ester, L, L-APM). The Joint FAO/WHO Expert Committee on Food Additives has set 0.04 wt% as the maximum permitted level of the sum of L, D-APM and D, L-APM in commercially available L, L-APM. In this study, we developed and validated a simple high-performance liquid chromatography (HPLC) method using an ODS column to determine L, D-APM and D, L-APM in L, L-APM. The limits of detection and quantification, respectively, of L, D-APM and D, L-APM were found to be 0.0012 wt% and 0.004 wt%. This method gave excellent accuracy, repeatability, and reproducibility in a recovery test performed on five different days. Moreover, the method was successfully applied to the determination of these diastereomers in commercial L, L-APM samples. Thus, the developed method is a simple, useful, and practical tool for determining L, D-APM and D, L-APM levels in L, L-APM.     

Characterization of Age-Dependent and Progressive Cortical Neuronal Degeneration in Presenilin Conditional Mutant Mice

Presenilins are the major causative genes of familial Alzheimer''s disease (AD). Our previous study has demonstrated essential roles of presenilins in memory and neuronal survival. Here, we explore further how loss of presenilins results in age-related, progressive neurodegeneration in the adult cerebral cortex, where the pathogenesis of AD occurs. To circumvent the requirement of presenilins for embryonic development, we used presenilin conditional double knockout (Psen cDKO) mice, in which presenilin inactivation is restricted temporally and spatially to excitatory neurons of the postnatal forebrain beginning at 4 weeks of age. Increases in the number of degenerating (Fluoro-Jade B+, 7.6-fold) and apoptotic (TUNEL+, 7.4-fold) neurons, which represent ∼0.1% of all cortical neurons, were first detected at 2 months of age when there is still no significant loss of cortical neurons and volume in Psen cDKO mice. By 4 months of age, significant loss of cortical neurons (∼9%) and gliosis was found in Psen cDKO mice. The apoptotic cell death is associated with caspase activation, as shown by increased numbers of cells immunoreactive for active caspases 9 and 3 in the Psen cDKO cortex. The vulnerability of cortical neurons to loss of presenilins is region-specific with cortical neurons in the lateral cortex most susceptible. Compared to the neocortex, the increase in apoptotic cell death and the extent of neurodegeneration are less dramatic in the Psen cDKO hippocampus, possibly in part due to increased neurogenesis in the aging dentate gyrus. Neurodegeneration is also accompanied with mitochondrial defects, as indicated by reduced mitochondrial density and altered mitochondrial size distribution in aging Psen cortical neurons. Together, our findings show that loss of presenilins in cortical neurons causes apoptotic cell death occurring in a very small percentage of neurons, which accumulates over time and leads to substantial loss of cortical neurons in the aging brain. The low occurrence and significant delay of apoptosis among cortical neurons lacking presenilins suggest that loss of presenilins may induce apoptotic neuronal death through disruption of cellular homeostasis rather than direct activation of apoptosis pathways.     

Comparison of Proteins Involved with Cyclic AMP Metabolism Between Synaptic Membrane and Postsynaptic Density Preparations Isolated from Canine Cerebral Cortex and Cerebellum

Synaptic membrane and postsynaptic density (PSD) fractions isolated from canine cerebral cortex and cerebellum were assayed for the following proteins: adenylate cyclase and phosphodiesterase (PDE) activities against cyclic AMP and cyclic GMP, the regulatory subunit of the cyclic AMP-dependent protein kinase, and the substrate proteins for this kinase. The results were expressed on the basis of both the protein content of the fractions and the number of synapses in the synaptic membrane fractions. The number of synapses on a constant protein content basis was about three times higher in the cerebral cortex synaptic membrane fraction than in the comparable cerebellar fraction. Adenylate cyclase activity was from 3.4 to 5.6 times higher in the cerebral cortex membrane fraction than in the cerebellar membrane fraction based on protein content but only slightly higher based on synapse counts. PSD fractions had no adenylate cyclase activity. The cyclic AMP-PDE activity was from 17 to 27 times higher in the cerebral cortex membrane fraction than in the cerebellar membrane fraction based on protein content, and about five times higher based on synapse counts. By doing PDE histochemistry at the electron microscopy level it was found that all the cerebral cortex PSDs in the isolated fraction contained PDE activity, none being found associated with the broken-up material in the fraction. The amount of the regulatory subunit of the cyclic AMP-dependent protein kinase was about equal in the two fractions based on protein, but about one-third lower in cerebral cortex fraction than in cerebellar fractions. In the cerebral cortex membrane fraction the primary substrate for the cyclic AMP-dependent protein kinase is synapsin I, with much lower amounts in the cerebellar membrane fraction. The PSD fraction from the two sources also showed these differences in synapsin I content. In the cerebellar membrane fraction, the primary substrate for the enzyme is a approximately 245,000 Mr protein not found in the cerebral cortex membrane fraction. The findings that the turnover of cyclic AMP is much higher in cerebral cortex synapses than in cerebellar synapses, and that differences are found between the cerebral cortex and cerebellum with regard to the substrate proteins for the cyclic AMP-dependent protein kinase indicate a divergence in the effect of cyclic AMP between cerebral cortex and cerebellar synapses.     

Rice chitinases: sugar recognition specificities of the individual subsites

Sugar recognition specificities of class III (OsChib1a) and class I (OsChia1cDeltaChBD) chitinases from rice, Oryza sativa L., were investigated by analyzing (1)H- and (13)C-nuclear magnetic resonance spectra of the enzymatic products from partially N-acetylated chitosans. The reducing end residue of the enzymatic products obtained by the class III enzyme was found to be exclusively acetylated, whereas both acetylated and deacetylated units were found at the nearest neighbor to the reducing end residue. Both acetylated and deacetylated units were also found at the nonreducing end residue and its nearest neighbor of the class III enzyme products. Thus, only subsite (-1) among the contiguous subsites (-2) to (+2) of the class III enzyme was found to be specific to an acetylated residue. For the class I enzyme, the reducing end residue was preferentially acetylated, although the specificity was not absolute. The nearest neighbor to the acetylated reducing end residue was specifically acetylated. Moreover, the nonreducing end residue produced by the class I enzyme was exclusively acetylated, although there was a low but significant preference for deacetylated units at the nearest neighbor to the nonreducing end. These results suggest that the three contiguous subsites (-2), (-1), and (+1) of the class I enzyme are specific to three consecutive GlcNAc residues of the substrate. In rice plants, the target of the class I enzyme might be a consecutive GlcNAc sequence probably in the cell wall of fungal pathogen, whereas the class III enzyme might act toward an endogenous complex carbohydrate containing GlcNAc residue.     

Development of cysteine-free fluorescent proteins for the oxidative environment

Molecular imaging employing fluorescent proteins has been widely used to highlight specific reactions or processes in various fields of the life sciences. Despite extensive improvements of the fluorescent tag, this technology is still limited in the study of molecular events in the extracellular milieu. This is partly due to the presence of cysteine in the fluorescent proteins. These proteins almost cotranslationally form disulfide bonded oligomers when expressed in the endoplasmic reticulum (ER). Although single molecule photobleaching analysis showed that these oligomers were not fluorescent, the fluorescent monomer form often showed aberrant behavior in folding and motion, particularly when fused to cysteine-containing cargo. Therefore we investigated whether it was possible to eliminate the cysteine without losing the brightness. By site-saturated mutagenesis, we found that the cysteine residues in fluorescent proteins could be replaced with specific alternatives while still retaining their brightness. cf(cysteine-free)SGFP2 showed significantly reduced restriction of free diffusion in the ER and marked improvement of maturation when fused to the prion protein. We further applied this approach to TagRFP family proteins and found a set of mutations that obtains the same level of brightness as the cysteine-containing proteins. The approach used in this study to generate new cysteine-free fluorescent tags should expand the application of molecular imaging to the extracellular milieu and facilitate its usage in medicine and biotechnology.     

Biospecific fractionation of chitosan

We have previously reported that, although a fully de-N-acetylated chitosan does not bind to hen egg white lysozyme, chitosans with a low fraction of N-acetylated units (FA) bind biospecifically to lysozyme with an affinity strongly dependent upon pH and ionic strength and without concomitant cleavage of glycosidic linkages. In this study, we report on the fractionation of a low FA chitosan with low molecular weight by biospecific adsorption of the chitosan molecules containing N-acetyl groups to immobilized lysozyme. Hen egg white lysozyme was immobilized to CNBr-activated Sepharose 4B, and a chitosan with a fraction of N-acetylated units of 0.045 and a weight average degree of polymerization (DPw) of 22 was applied to the column at suitable conditions for biospecific binding (pH 5.7, 0.15 M NaCl). The chitosan could be separated into two fractions, one that was not adsorbed to the lysozyme-column and one that was adsorbed and could be eluted by decreasing the pH and the ionic strength (0.08 M acetic acid of pH 3.0). The fractions were analyzed and the fraction that was not adsorbed was found to be fully de-N-acetylated chitosan with a DPw of 18, whereas the fraction that was adsorbed was a chitosan with FA of 0.080 and DPw of 24. Experimental data were compared with data from theoretical calculations, which was used to calculate the fraction of chitosan molecules with and without acetyl groups, showing good correlation between experimental and theoretical results.     

Comparative study of the reaction mechanism of family 18 chitinases from plants and microbes

Hydrolytic mechanisms of family 18 chitinases from rice (Oryza sativa L.) and Bacillus circulans WL-12 were comparatively studied by a combination of HPLC analysis of the reaction products and theoretical calculation of reaction time-courses. All of the enzymes tested produced beta-anomers from chitin hexasaccharide [(GlcNAc)(6)], indicating that they catalyze the hydrolysis through a retaining mechanism. The rice chitinases hydrolyzed predominantly the fourth and fifth glycosidic linkages from the nonreducing end of (GlcNAc)(6), whereas B. circulans chitinase A1 hydrolyzed the second linkage from the nonreducing end. In addition, the Bacillus enzyme efficiently catalyzed transglycosylation, producing significant amounts of chitin oligomers larger than the initial substrate, but the rice chitinases did not. The time-courses of (GlcNAc)(6) degradation obtained by HPLC were analyzed by theoretical calculation, and the subsite structures of the rice chitinases were identified to be (-4)(-3)(-2)(-1)(+1)(+2). From the HPLC profile of the reaction products previously reported [Terwisscha van Scheltinga et al. (1995) Biochemistry 34, 15619-15623], family 18 chitinase from rubber tree (Hevea brasiliensis) was estimated to have the same type of subsite structure. Theoretical analysis of the reaction time-course for the Bacillus enzyme revealed that the enzyme has (-2)(-1) (+1)(+2)(+3)(+4)-type subsite structure, which is identical to that of fungal chitinase from Coccidioides immitis [Fukamizo et al. (2001) Biochemistry 40, 2448-2454]. The Bacillus enzyme also resembled the fungal chitinase in its transglycosylation activity. Minor structural differences between plant and microbial enzymes appear to result in such functional variations, even though all of these chitinases are classified into the identical family of glycosyl hydrolases.