Efficient immobilization of enzymes on microchannel surface through His-tag and application for microreactor

We developed a simple immobilisation method for His-tagged enzymes on a microchannel surface. It facilitates immobilisation of protein molecule on microchannel surface through Ni-complex, using crude or purified protein solutions. By this method, we could immobilize proteins on microcapillary constantly. This method might be useful for further development of microreactor with reversibly immobilized enzymes.     

Taste cell responses in the frog are modulated by parasympathetic efferent nerve fibers

We studied the anatomical properties of parasympathetic postganglionic neurons in the frog tongue and their modulatory effects on taste cell responses. Most of the parasympathetic ganglion cell bodies in the tongue were found in extremely small nerve bundles running near the fungiform papillae, which originate from the lingual branches of the glossopharyngeal (GP) nerve. The density of parasympathetic postganglionic neurons in the tongue was 8000-11,000/mm(3) of the extremely small nerve bundle. The mean major axis of parasympathetic ganglion cell bodies was 21 microm, and the mean length of parasympathetic postganglionic neurons was 1.45 mm. Electrical stimulation at 30 Hz of either the GP nerve or the papillary nerve produced slow hyperpolarizing potentials (HPs) in taste cells. After nicotinic acetyl choline receptors on the parasympathetic ganglion cells in the tongue had been blocked by intravenous (i.v.) injection of D-tubocurarine (1 mg/kg), stimulation of the GP nerve did not induce any slow HPs in taste cells but that of the papillary nerve did. A further i.v. injection of a substance P NK-1 antagonist, L-703,606, blocked the slow HPs induced by the papillary nerve stimulation. This suggests that the parasympathetic postganglionic efferent fibers innervate taste cells and are related to a generation of the slow HPs and that substance P is released from the parasympathetic postganglionic axon terminals. When the resting membrane potential of a taste cell was hyperpolarized by a prolonged slow HP, the gustatory receptor potentials for NaCl and sugar stimuli were enhanced in amplitude, but those for quinine-HCl and acetic acid stimuli remained unchanged. It is concluded that frog taste cell responses are modulated by activities of parasympathetic postganglionic efferent fibers innervating these cells.     

Identification of specific [3H]adenine-binding sites in rat brain membranes

We recently reported that adenine acts as a neurotrophic factor independent of adenosine or P2 receptors in cultured Purkinje cells [Watanabe S. et al. (2003) J. Neurosci. Res. 74, 754-759], suggesting the presence of specific receptors for adenine in the brain. In this study, the characterization of adenine-binding activity in the rat brain was performed to further characterize the receptor-like adenine-binding sites. Specific binding sites for [(3)H]adenine were detected in membrane fractions prepared from rat brains. The kinetics of [(3)H]adenine binding to membranes was described by the association and dissociation rate constants, 8.6 x 10(5) M(-1) min(-1) and 0.118 +/- 0.045 min(-1), respectively. A single binding site for [(3)H]adenine with a K (D) of 157.1 +/- 20.8 nM and a B (max) of 16.3 +/- 1.1 pmol/mg protein (n = 6) was demonstrated in saturation experiments. A displacement study involving various related compounds showed that the [(3)H]adenine binding was highly specific for adenine. It was also found that [(3)H]adenine-binding activity was inhibited by adenosine, although other adenosine receptor ligands were ineffective as to [(3)H]adenine binding. The brain, especially the cerebellum and spinal cord, showed the highest [(3)H]adenine-binding activity of the tissues examined. These results are consistent with the presence of a novel adenine receptor in rat brain membranes.     

beta Subunits of voltage-gated sodium channels are novel substrates of beta-site amyloid precursor protein-cleaving enzyme (BACE1) and gamma-secretase

Sequential processing of amyloid precursor protein (APP) by membrane-bound proteases, BACE1 and gamma-secretase, plays a crucial role in the pathogenesis of Alzheimer disease. Much has been discovered on the properties of these proteases; however, regulatory mechanisms of enzyme-substrate interaction in neurons and their involvement in pathological changes are still not fully understood. It is mainly because of the membrane-associated cleavage of these proteases and the lack of information on new substrates processed in a similar way to APP. Here, using RNA interference-mediated BACE1 knockdown, mouse embryonic fibroblasts that are deficient in either BACE1 or presenilins, and BACE1-deficient mouse brain, we show clear evidence that beta subunits of voltage-gated sodium channels are sequentially processed by BACE1 and gamma-secretase. These results may provide new insights into the underlying pathology of Alzheimer disease.     

Small GTPase proteins Rin and Rit Bind to PAR6 GTP-dependently and regulate cell transformation

The novel small GTPases Rin and Rit are close relatives of Ras, and recent studies show that they play a role in mediating neuronal differentiation. However, the direct effectors of Rin and Rit have yet to be fully characterized. Here we showed that Rin and Rit directly bind to the PDZ domain of PAR6, a cell polarity-regulating protein, in a GTP-dependent manner both in vivo and in vitro. Moreover, Rin and Rit can form a ternary complex consisting of PAR6 and Rac/Cdc42, members of the Rho family of small GTPases modulating cell growth and polarity. This ternary complex synergistically potentiates cell transformation in NIH3T3 cells, and the interaction between Rin/Rit and the PDZ domain of PAR6 is important for this effect. These results suggest that the Rin/Rit-PAR6-Rac/Cdc42 ternary complex may work physiologically in the cells, such as in tumorigenesis.     

An increase in circulating mast cell colony-forming cells in asthma

We compared a potential to generate mast cells among various sources of CD34(+) peripheral blood (PB) cells in the presence of stem cell factor (SCF) with or without thrombopoietin (TPO), using a serum-deprived liquid culture system. From the time course of relative numbers of tryptase-positive and chymase-positive cells in the cultured cells grown by CD34(+) PB cells of nonasthmatic healthy individuals treated with G-CSF, TPO appears to potentiate the SCF-dependent growth of mast cells without influencing the differentiation into mast cell lineage. CD34(+) PB cells from asthmatic patients in a stable condition generated significantly more mast cells under stimulation with SCF alone or SCF+TPO at 6 wk of culture than did steady-state CD34(+) PB cells of normal controls. Single-cell culture studies showed a substantial difference in the number of SCF-responsive or SCF+TPO-responsive mast cell progenitors in CD34(+) PB cells between the two groups. In the presence of TPO, CD34(+) PB cells from asthmatic children could respond to a suboptimal concentration of SCF to a greater extent, compared with the values obtained by those of normal controls. Six-week cultured mast cells of asthmatic subjects had maturation properties (intracellular histamine content and tryptase/chymase enzymatic activities) similar to those derived from mobilized CD34(+) PB cells of nonasthmatic subjects. An increase in a potential of circulating hemopoietic progenitors to differentiate into mast cell lineage may contribute to the recruitment of mast cells toward sites of asthmatic mucosal inflammation.     

Neuroprotective effects of non-steroidal anti-inflammatory drugs by direct scavenging of nitric oxide radicals

Recently, it has been reported that inflammatory processes are associated with the pathophysiology of Alzheimer's disease and that treatment of non-steroidal anti-inflammatory drugs reduce the risk for Alzheimer's disease. In the present study, we examined nitric oxide radical quenching activity of non-steroidal anti-inflammatory drugs and steroidal drugs using our established direct in vitro nitric oxide radical detecting system by electron spin resonance spectrometry. The non-steroidal anti-inflammatory drugs, aspirin, mefenamic acid, indomethacin and ketoprofen directly and dose-dependently scavenged generated nitric oxide radicals. In experiments of nitric oxide radical donor, NOC18-induced neuronal damage, these four non-steroidal drugs significantly prevented the NOC18-induced reduction of cell viability and apoptotic nuclear changes in neuronal cells without affecting the induction of inducible nitric oxide synthase-like immunoreactivity. However, ibuprofen, naproxen or steroidal drugs, which had less or no scavenging effects in vitro, showed almost no protective effects against NOC18-induced cell toxicity. These results suggest that the protective effects of the former four non-steroidal anti-inflammatory drugs against apoptosis might be mainly due to their direct nitric oxide radical scavenging activities in neuronal cells. These direct NO. quenching activities represent novel effects of non-steroidal anti-inflammatory drugs. Our findings identified novel pharmacological mechanisms of these drugs to exert not only their anti-inflammatory, analgesic, antipyretic activities but also neuroprotective activities against neurodegeneration.     

Activity of a sperm-borne oocyte-activating factor in spermatozoa and spermatogenic cells from cynomolgus monkeys and its localization after oocyte activation

It is widely accepted that mature mammalian oocytes are induced to resume meiosis by a sperm-borne oocyte-activating factor(s) (sperm factor, SF) immediately after normal fertilization or intracytoplasmic sperm injection. The SF is most likely a soluble factor that is localized within the cytoplasm of mature spermatozoa, but the exact stage at which it appears during spermatogenesis and its localization after oocyte activation is not fully understood, except in the mouse. First, we injected mature spermatozoa and spermatogenic cells from cynomolgus monkeys into mouse oocytes to assess their oocyte-activating capacity. More than 90% of mouse oocytes were activated after injection of monkey spermatozoa. Round spermatids and primary spermatocytes (late pachytene to diplotene) also activated oocytes (93% and 79%, respectively). Injection of monkey spermatozoa and spermatids induces intracellular Ca(2+) oscillations in a pattern similar to that seen following normal fertilization. Most spermatocytes did not produce typical intracellular Ca(2+) oscillations. Second, we transferred pronuclei or cytoplasts from mouse oocytes that had been activated by monkey spermatozoa or spermatids into intact mature mouse oocytes by electrofusion in order to examine the localization of the SF after pronuclear formation. Some of the SF was localized within the pronuclei, but some stayed in the ooplasm. This study demonstrated that spermatogenic cells of cynomolgus monkeys acquire oocyte-activating capacity at much earlier stages than those of mice, and that the monkey SF has a pronucleus-directing nature, although to a lesser extent than the mouse SF.     

NHE6 protein possesses a signal peptide destined for endoplasmic reticulum membrane and localizes in secretory organelles of the cell

The NHE6 protein is a unique Na(+)/H(+) exchanger isoform believed to localize in mitochondria. It possesses a hydrophilic N-terminal portion that is rich in positively charged residues and many hydrophobic segments. In the present study, signal sequences in the NHE6 molecule were examined for organelle localization and membrane topogenesis. When the full-length protein was expressed in COS7 cells, it localized in the endoplasmic reticulum and on the cell surface. Furthermore, the protein was fully N-glycosylated. When green fluorescent protein was fused after the second (H2) or third (H3) hydrophobic segment, the fusion proteins were targeted to the endoplasmic reticulum (ER) membrane. The localization pattern was the same as that of fusion proteins in which green fluorescent protein was fused after H2 of NHE1. In an in vitro system, H1 behaved as a signal peptide that directs the translocation of the following polypeptide chain and is then processed off. The next hydrophobic segment (H2) halted translocation and eventually became a transmembrane segment. The N-terminal hydrophobic segment (H1) of NHE1 also behaved as a signal peptide. Cell fractionation studies using antibodies against the 15 C-terminal residues indicated that NHE6 protein localized in the microsomal membranes of rat liver cells. All of the NHE6 molecules in liver tissue possess an endoglycosidase H-resistant sugar chain. These findings indicate that NHE6 protein is targeted to the ER membrane via the N-terminal signal peptide and is sorted to organelle membranes derived from the ER membrane.