Endosomal acidification by Na+/H+ exchanger NHE5 regulates TrkA cell-surface targeting and NGF-induced PI3K signaling

To facilitate polarized vesicular trafficking and signal transduction, neuronal endosomes have evolved sophisticated mechanisms for pH homeostasis. NHE5 is a member of the Na⁺/H⁺ exchanger family and is abundantly expressed in neurons and associates with recycling endosomes. Here we show that NHE5 potently acidifies recycling endosomes in PC12 cells. NHE5 depletion by plasmid-based short hairpin RNA significantly reduces cell surface abundance of TrkA, an effect similar to that observed after treatment with the V-ATPase inhibitor bafilomycin. A series of cell-surface biotinylation experiments suggests that anterograde trafficking of TrkA from recycling endosomes to plasma membrane is the likeliest target affected by NHE5 depletion. NHE5 knockdown reduces phosphorylation of Akt and Erk1/2 and impairs neurite outgrowth in response to nerve growth factor (NGF) treatment. Of interest, although both phosphoinositide 3-kinase–Akt and Erk signaling are activated by NGF-TrkA, NGF-induced Akt-phosphorylation appears to be more sensitively affected by perturbed endosomal pH. Furthermore, NHE5 depletion in rat cortical neurons in primary culture also inhibits neurite formation. These results collectively suggest that endosomal pH modulates trafficking of Trk-family receptor tyrosine kinases, neurotrophin signaling, and possibly neuronal differentiation.     

Polysaccharide-polynucleotide complexes (15): thermal stability of schizophyllan (SPG)/poly(C) triple strands is controllable by alpha-amino acid modification

Schizophyllan (SPG), a beta-1,3-glucan polysaccharide which is known to form macromolecular complexes with certain polynucleotides, was modified by a reductive amination method with alpha-amino acids (Arg, Lys, and Ser). The thermal stability of the complexes as estimated by T(m) was enhanced in SPG-Arg and SPG-Lys conjugates which have pI values higher than the pH of the medium (8.0). The T(m) shift increased with the increase in the percentage of alpha-amino acid introduced and the highest T(m) values attained were 64 degrees C for SPG-Arg conjugate and 62 degrees C for SPG-Lys conjugate, which are higher by 13 and 11 degrees C, respectively, than those of the unmodified SPG+poly(C) complex. In the SPG-Ser conjugate with a pI lower than the medium pH (8.0), the T(m) values decreased with an increase in the percentage of Ser. Formation of the macromolecular complex was no longer detected above 13.2% Ser. The findings indicate that the T(m) values are easily controllable by the type and percentage of the introduced alpha-amino acids. We believe, therefore, that the present conjugates, consisting of naturally originated SPG and alpha-amino acids, provide an important lead for developing nontoxic artificial vectors and to control the affinity with polynucleotides in response to medium pH and temperature.     

Direct atomization atomic absorption spectrometry for intact biological samples: application to trace metal analysis in silkworm eggs and their hatched worms.

Highly sensitive carbon rod atomic absorption for trace elements was observed by direct atomization of samples with no pretreatment for intact silkworm eggs, their hatched worms, or National Bureau of Standards biological samples (orchard leaves and bovine liver). Crucial temperatures for drying, ashing, and atomization processes were found to obtain accurate results. Results were consistent with those of activation analyses and of ordinary atomic absorption with wet ashing. The precision and dynamic range of this method were evaluated. The advantages and disadvantages of the present approach were critically discussed.     

Interleukin-13 induces thymus and activation-regulated chemokine (CCL17) in human peripheral blood mononuclear cells

BACKGROUND: In allergic inflammation involving allergic rhinitis, the predominance of Th(2) lymphocytes is one of the primary causal agents in promotion of the allergic condition. Thymus and activation-regulated chemokine (TARC/CCL17) is a recently identified chemokine that induces the development of Th(2) lymphocytes. One of the sources of TARC has been reported to be peripheral blood mononuclear cells (PBMCs). OBJECTIVE: We investigated TARC production from PBMCs by the stimulation of specific antigens and Th(2) type cytokines. METHOD: PBMCs were isolated from both allergic rhinitis patients and healthy volunteers. PBMCs were incubated with cytokine. TARC mRNA expression was examined by real time PCR methods and the amount of TARC production was examined by ELISA. RESULTS: IL-13 was found to be the most potent inducer for TARC mRNA expression and protein production in PBMCs. Furthermore, tumour necrosis factor alpha and IL-13 synergistically induce TARC. The amount of TARC from allergic rhinitis patients was significantly larger than that from healthy volunteers. Moreover, TARC was induced by a specific antigen, and was 35% inhibited by an anti-IL-13 neutralizing antibody. CONCLUSION: These results indicate that IL-13 is important in TARC mediated Th(2) lymphocytes infiltration in the nasal mucosa.     

Effects of melanin on tyrosine hydroxylase and phenylalanine hydroxylase.

Melanin inhibited rat liver phenylalanine hydroxylase, but activated tyrosine hydroxylase from rat brain (caudate nucleus), rat adrenal glands, and bovine adrenal medulla. Activation of tyrosine hydroxylase by melanin was demonstrated with the extensively dialyzed enzyme and in suboptimal concentrations of the substrate (tyrosine) and the cofactor (6-methyltetrahydropterin). Tyrosine hydroxylase from rat brain was activated by melanin more markedly than that from rat adrenal glands. Purified and extensively dialyzed bovine adrenal tyrosine hydroxylase had two Km values with 6-methyltetrahydropterin, depending upon its concentrations, but the melanin-activated tyrosine hydroxylase had a single Km value and showed the classical Michaelis-Menten kinetics.     

'Click chemistry' on polysaccharides: a convenient, general, and monitorable approach to develop (1-->3)-beta-D-glucans with various functional appendages

(1-->3)-beta-D-Glucans having various functional appendages (lactoside, ferrocene, pyrene, and porphyrin) can be prepared in an convenient, quantitative, and regioselective manner through regioselective bromination-azidation of curdlan to afford 6-azido-6-deoxycurdlan followed by chemoselective [3+2]-cycloadditions with various functional modules bearing a terminal alkyne group. The ability to monitor reaction conversions is an additional advantage of this synthetic approach over the conventional direct modifications on polysaccharides; the reaction can be readily monitored based on the intensity of azido peaks in the in situ attenuated total reflection infrared spectra.