The effects of doxapram on medullary respiratory neurones in brainstem-spinal cord preparations from newborn rats.

Doxapram is the only dedicated respiratory stimulant used to aid recovery of breathing after major surgery. Doxapram acts on peripheral chemoreceptors and although the central action of doxapram has been suggested, its detailed neuronal mechanism is unknown. We assessed doxapram-induced changes in spontaneous cervical nerve (C4) inspiratory activity and the firing of action potentials in pre-inspiratory and inspiratory neurones in the medulla. Experiments were performed in neonatal rat brainstem-spinal cord preparations, which can produce respiratory rhythm for several hours under in vitro conditions. Doxapram application (for 15 min) increased the frequency and amplitude of C4 activity dose-dependently. Doxapram induced changes in the electrophysiological properties of pre-inspiratory and inspiratory neurones. Our results suggest that respiratory activity enhancement was likely to be induced via effects on the potassium channels of pre-inspiratory and inspiratory neurones and indicate the central actions of doxapram.

Glycogen synthase kinase-3beta and p38 phosphorylate cyclin D2 on Thr280 to trigger its ubiquitin/proteasome-dependent degradation in hematopoietic cells.

Cyclin D2 plays an important role in regulation of hematopoietic cell proliferation by cytokines and is implicated in oncogenesis of various hematopoietic malignancies. However, mechanisms regulating cyclin D2 stability and its expression level have remained to be known. Here, we demonstrate that interleukin-3 signaling stabilizes cyclin D2 by inhibition of glycogen synthase kinase-3beta (GSK3beta) through Janus kinase2-dependent activation of phosphatidylinositol 3'-kinase (PI3K)/Akt signaling pathway in hematopoietic 32Dcl3 cells. On the other hand, osmotic stress was shown to induce a rapid proteasomal degradation of cyclin D2, which was mediated by activation of p38. GSK3beta and p38 was demonstrated to phosphorylate cyclin D2 on Thr280 in vitro, while a cyclin D2 mutant with this residue substituted with Ala was found to be resistant to ubiquitination and proteasome-dependent degradation in 32Dcl3 cells. Inhibition of the PI3K pathway or induction of osmotic stress also caused a rapid proteasomal degradation of cyclin D2 in primary leukemic or myeloma cells. These results indicate that cyclin D2 expression in normal and malignant hematopoietic cells is regulated by ubiquitin/proteasome-dependent degradation that is triggered by Thr280 phosphorylation by GSK3beta or p38, which is induced by inhibition of the PI3K pathway or by osmotic stress, respectively.

Pharmacological evidence of cholinergic involvement in adult hippocampal neurogenesis in rats.

In adult hippocampus, neural progenitor cells give rise to neurons throughout life, and the neurogenesis is modulated by various intrinsic and extrinsic factors. Recent reports showed that lesion of septal cholinergic nuclei projecting to hippocampus suppressed the survival of newborn cells in the dentate gyrus (DG) of hippocampus. Here, we studied whether pharmacological treatment to activate or inhibit the cholinergic system could modulate adult hippocampal neurogenesis. 5'-Bromo-2'-deoxyuridine (BrdU) was injected to label dividing cells before the drug treatment. Immunohistochemical analysis was performed in normal rats chronically treated with an acetylcholinesterase inhibitor donepezil or a muscarinic acetylcholine receptor blocker scopolamine for four weeks. Donepezil increased, but scopolamine decreased, the number of BrdU-positive cells in the DG as compared with the control. Neither drug altered the percentage of BrdU-positive cells that were also positive for a neuronal marker neuronal nuclei, nor net population of proliferative cells labeled with proliferating cell nuclear antigen. We also found that donepezil enhanced, and scopolamine suppressed, the expression level of phosphorylated cAMP response element binding protein (CREB), which is related to cell survival, in the DG. These results indicate that donepezil enhances and scopolamine suppresses the survival of newborn cells in the DG via CREB signaling without affecting neural progenitor cell proliferation and the neuronal differentiation. This is the first evidence that pharmacological manipulation of the cholinergic system can modulate adult hippocampal neurogenesis.

Hippocampal Damage Disrupts Eyeblink Conditioning in Mice Lacking Glutamate Receptor Subunit δ2.

Cerebellar long-term depression (LTD) at the parallel fiber-Purkinje cell synapses has been proposed to be a neural substrate for classical eyeblink conditioning. Mutant mice lacking the glutamate receptor subunit δ2 (GluRδ2), in which the cerebellar LTD is disrupted, exhibited a severe impairment in the delay eyeblink conditioning with a temporal overlap of CS and US. However, they learned normally trace and delay conditioning without CS-US overlap, suggesting a learning mechanism which does not require the cerebellar LTD.In the present study, we tested possible involvement of the hippocampus in this cerebellar LTD-independent learning. We examined effects of scopolamine and hippocampal lesion on the delay conditioning without CS-US overlap. TheGluRδ2 mutant mice that received scopolamine or aspiration of the dorsalhippocampus together with its overlying cortex exhibited a severe impairment in learning, while the control mutant mice that received saline or aspiration of the overlying cortex learned normally. In contrast, wild-type mice that received either treatment learned as normally as the control wild-type mice. These results suggest that the hippocampus is essential in the cerebellar LTD-independent learning in the GluRδ2 mutant mice, indicating a newrole of hippocampus in the paradigm with a short trace interval.

Activation of Cdh1-dependent APC is required for G1 cell cycle arrest and DNA damage-induced G2 checkpoint in vertebrate cells.

Anaphase-promoting complex (APC) is activated by two regulatory proteins, Cdc20 and Cdh1. In yeast and Drosophila, Cdh1-dependent APC (Cdh1-APC) activity targets mitotic cyclins from the end of mitosis to the G1 phase. To investigate the function of Cdh1 in vertebrate cells, we generated clones of chicken DT40 cells disrupted in their Cdh1 loci. Cdh1 was dispensable for viability and cell cycle progression. However, similarly to yeast and Drosophila, loss of Cdh1 induced unscheduled accumulation of mitotic cyclins in G1, resulting in abrogation of G1 arrest caused by treatment with rapamycin, an inducer of p27(Kip1). Further more, we found that Cdh1(-/-) cells fail to maintain DNA damage-induced G2 arrest and that Cdh1-APC is activated by X-irradiation-induced DNA damage. Thus, activation of Cdh1-APC plays a crucial role in both cdk inhibitor-dependent G1 arrest and DNA damage-induced G2 arrest.

Hyperpolarizing shift by quinine in the steady-state inactivation curve of delayed rectifier-type potassium current in bullfrog sympathetic neurons.

Whole-cell recordings were made from dissociated bullfrog sympathetic neurons to examine the actions of quinine (1-100 microM) on the steady-state activation and inactivation curves of a delayed rectifier-type potassium current (I(K)). Quinine (EC50 approximately 8 microM) caused a hyperpolarizing shift (approximately 31 mV with 30 microM) in the inactivation curve of I(K) without significantly affecting its activation curve. Quinine (20 microM) was without effects on the voltage-dependence of a rapidly-inactivating A-type potassium current (I(A)). It is concluded that quinine can selectively modulate the voltage-dependence of I(K) in amphibian autonomic neurons.

The actin-depolymerizing factor destrin has an actin-stabilizing domain.

Destrin is a 19 kDa actin-depolymerizing protein of the ADF-cofilin family. Destrin was digested with trypsin to a structurally stable 9.2 kDa fragment that contains the actin-binding sequence. The purified 9.2 kDa fragment has an actin filament stabilizing activity, rather than an actin filament depolymerizing activity. The deleted region is probably essential for the actin filament depolymerizing activity of intact destrin. Surprisingly, the 9.2 kDa fragment also has an assembly-promoting activity in the absence of ATP.

Purification and characterization of a new, ubiquitously distributed class of microtubule-associated protein with molecular mass 250 kDa.

A heat-stable microtubule-associated protein (MAP) with relative molecular mass 250 000, termed 250-kDa MAP, was purified from bovine adrenal cortex. It is classified as a MAP subspecies distinct from MAP1, MAP2, tau, and MAP4, as judged from its electrophoretic mobility, heat stability and immunoreactivity. Purified 250-kDa MAP was able to bind to taxol-stabilized microtubules, although it lacked the ability to polymerize purified tubulin into microtubules. Western-blot analysis showed that this MAP was expressed ubiquitously in mammalian tissues. Immunofluorescence microscopy revealed that polyclonal antibodies raised against 250-kDa MAP stained many punctate structures in the cytoplasm of cultured cells. Blurry cytosolic staining was also observed. Judging from the result of nocodazole treatment, the punctate structures were associated with the microtubule network throughout the cytoplasm, while cytosolic 250-kDa MAP colocalized with free tubulin. Under electron microscopy, 250-kDa MAP has the appearance of a hollow sphere of about 12 nm diameter.

Effects of lanthanides on voltage-dependent potassium currents in bullfrog sympathetic neurons.

The effects of lanthanides (La(3+), Gd(3+), Lu(3+) and Sm(3+)) on voltage-dependent potassium currents were studied in dissociated bullfrog sympathetic neurons. A-type current (I(A)) and M-type current (I(M)) were blocked by lanthanides (0.1-30 microM) with I(M) being much less sensitive to these ions than I(A). The order of potency was Gd(3+)>/=Lu(3+) approximately La(3+) approximately Sm(3+) for I(A) and Gd(3+)&z.Gt;Lu(3+) approximately La(3+)>Sm(3+) for I(M). The I(M) block occurred independently of its activation kinetics while the I(A) block was associated with a positive shift of the activation and inactivation curves. Gd(3+) (100 microM) blocked the delayed rectifier-type current (I(K)) by less than 20%; Lu(3+), La(3+) and Sm(3+) (100 microM for each) were without effect on I(K). It is concluded that I(A) was the most sensitive to lanthanides, and Gd(3+) was the most potent for all the currents in amphibian autonomic neurons.

Cytokine-inducing macromolecular glycolipids from Enterococcus hirae: improved method for separation and analysis of its effects on cellular activation.

Previously, we showed that several minor macromolecular glycolipids accounting for less than 5% of the lipoteichoic acid (LTA) fraction from Enterococcus hirae ATCC 9790 possess cytokine-inducing activity, whereas the purified LTA does not. In other words, the immunobiological activity of the LTA fraction reported in the 1980s was not attributable to LTA itself, but to other glycolipids coexisting in the fraction. In the present study, we improved the procedure of separation of the active glycolipids and evaluated their effects on cellular activation. The immunobiologically active glycolipids were separated from the crude glycolipid fraction obtained by hot phenol-water extraction of the cells. The total yield of active glycolipids was about fivefold higher than that separated by the previous method. Interleukin-6-inducing activities of the active glycolipids from 1,25-dihydroxy vitamin D(3)-differentiated human monocytic leukemia cells, THP-1, were inhibited by anti-CD14 mAbs in a dose-dependent manner. Macrophages from Toll-like receptor (TLR)-2-deficient or -4-deficient mice completely lacked the ability to produce tumor necrosis factor-alpha on stimulation with active glycolipids. These observations indicated that the cellular activation by the active glycolipids from E. hirae is mediated by CD14 and by both TLR2 and TLR4.

Mechanisms underlying the M-current block by barium in bullfrog sympathetic neurons.

Whole-cell/voltage-clamp recordings were made from dissociated bullfrog sympathetic neurons to examine the channel blocking actions of barium (3-2000 microM) on an M-type potassium current (I(M)). Barium (IC(50) approximately 105 microM) blocked I(M) without affecting the 50%-activation voltage ( approximately -35 mV) and the slope factor ( approximately 11 mV) of the activation curve. The results indicate that the barium block is independent of the kinetics of I(M).

A novel cytokine-inducing glycolipid isolated from the lipoteichoic acid fraction of Enterococcus hirae ATCC 9790: a fundamental structure of the hydrophilic part.

Previously, we showed that quantitatively minor several glycolipids totaling only less than 5% of the lipoteichoic acid (LTA) fraction from Enterococcus hirae ATCC 9790 possessed cytokine-inducing activity, whereas the major component (over 90%) did not [Suda et al. (1995) FEMS Immun Med Microbiol 12:97-112]. The major inactive component was shown to have the chemical structure as was proposed for the LTA by Fischer [Hashimoto et al. (1997) J Biochem 121:779-86], suggesting that so-called LTA is not a cytokine-inducing component in the Gram-positive bacteria. In the present paper, the structure of the hydrophilic part of one of the cytokine-inducing glycolipid tentatively named GL4 is elucidated. GL4 was first subjected to hydrolysis with aqueous HF to give a polysaccharide and a mixture of low molecular weight products. The polysaccharide was composed mainly of highly branching mannan as concluded from NMR and MS analyses of its acetolysis products. The low molecular weight products consisted of phosphate and glycerol, suggesting the presence of a poly(glycerophosphate) structure in the original GL4. From these observations, the hydrophilic part of GL4 was shown to consist of mannose-rich polysaccharide and poly(glycerophosphate), the latter being bound to the former by a phosphodiester linkage.

Microtubule-binding property of microtubule-associated protein 2 differs from that of microtubule-associated protein 4 and tau.

The microtubule-binding domains of microtubule-associated protein (MAP) 2, MAP4, and tau are structurally similar [Aizawa, H., Emori, Y., Murofushi, H., Kawasaki, H., Sakai., H., and Suzuki, K. (1990) J. Biol. Chem. 265, 13849-13855]. To compare the microtubule-binding mechanisms of the three MAPs, we performed a quantitative competition analysis using the three MAPs and the microtubule-binding domain fragment of MAP4 (PA4T fragment). The two-cycled microtubule protein fraction from bovine brain contains MAP1, MAP2, MAP4, and tau. When an excess of the PA4T fragment was added to the microtubule protein fraction, MAP4 and tau were completely released from the microtubules, while MAP1 remained bound. MAP2 was only partially released from the microtubules. The competition between MAP2 and MAP4 was further analyzed using purified MAP2, the PA4T fragment, and tubulin. About half of the MAP2 was still bound to the microtubules, even in the presence of an excess amount of the PA4T fragment. The microtubule-binding mechanisms of MAP2 and MAP4 seem to be different, in spite of their similar primary structures.

Regulation of APC activity by phosphorylation and regulatory factors.

Ubiquitin-dependent proteolysis of Cut2/Pds1 and Cyclin B is required for sister chromatid separation and exit from mitosis, respectively. Anaphase-promoting complex/cyclosome (APC) specifically ubiquitinates Cut2/Pds1 at metaphase-anaphase transition, and ubiquitinates Cyclin B in late mitosis and G1 phase. However, the exact regulatory mechanism of substrate-specific activation of mammalian APC with the right timing remains to be elucidated. We found that not only the binding of the activators Cdc20 and Cdh1 and the inhibitor Mad2 to APC, but also the phosphorylation of Cdc20 and Cdh1 by Cdc2-Cyclin B and that of APC by Polo-like kinase and cAMP-dependent protein kinase, regulate APC activity. The cooperation of the phosphorylation/dephosphorylation and the regulatory factors in regulation of APC activity may thus control the precise progression of mitosis.

A new model for microtubule-associated protein (MAP)-induced microtubule assembly. The Pro-rich region of MAP4 promotes nucleation of microtubule assembly in vitro.

The microtubule-binding domains of microtubule-associated protein (MAP) 2, tau, and MAP4 are divided into three distinctive regions: the Pro-rich region, the AP sequence region and the tail region (Aizawa, H., Emori, Y., Murofushi, H., Kawasaki, H., Sakai., H., and Suzuki, K. (1990) J. Biol. Chem. 265, 13849-13855). Electron microscopic observation showed that the taxol-stabilized microtubules alone and those mixed with the A4T fragment (containing the AP sequence region and the tail region) had a long, wavy appearance, while those mixed with the PA4T fragment (containing the Pro-rich region, the AP sequence region, and the tail region) or the PA4 fragment (containing the Pro-rich region and the AP sequence region) were shorter and straighter. Stoichiometries of the binding between the fragments and the tubulin dimers were approximately between 1 and 2, suggesting that not all of the AP sequences in the AP sequence region bound to tubulin. Binding affinity of the PA4T fragment is only four times higher than that of the A4T fragment, while the microtubule nucleating activity of the PA4T fragment is far greater. Based on these results, we propose that the nucleation of microtubule assembly is promoted by the bridging activity of the Pro-rich region in the MAPs.

Functional analysis of microtubule-binding domain of bovine MAP4.

Bovine microtubule-associated protein 4 (MAP4) consists of an amino-terminal projection domain and a carboxyl-terminal microtubule-binding domain. The carboxyl-terminal domain of MAP4 is further divided into three subdomains: a region rich in proline and basic residues (Pro-rich region), a region containing four repeats of an assembly-promoting (AP) sequence, which consists of 22 amino acid residues (AP sequence region), and a hydrophobic tail region (Tail region). The subdomain structure of MAP4 microtubule binding domain is similar to those of other MAPs (MAP2 and tau). In order to study the function of each subdomain per se of bovine MAP4 microtubule-binding domain, we purified a series of truncated fragments of MAP4, expressed in Escherichia coil. Binding affinity of the PA4T fragment (containing the Pro-rich region, the AP sequence region and the Tail region) is only four times higher than that of the A4T fragment (containing the AP sequence region and the Tail region), while the microtubule nucleating activity of the PA4T fragment is far greater. We propose that the Pro-rich region promotes the nucleation of microtubule assembly. The A4 fragment (corresponding to the AP sequence region) stimulated the assembly of tubulin into coldstable amorphous aggregates. The AP sequence region of MAP4 failed to promote microtubule assembly. On the other hand, the fragment has an activity to stimulate microtubule elongation. The function of the MAP4 Tail region is not clear at present. The A4T fragment (containing the AP sequence region and the Tail region) promote both microtubule nucleation and elongation step, but the A4 fragment only promotes microtubule elongation, suggesting that the Tail region is indispensable for the nucleation step. However, the fragment containing only the Tail region could not bind to microtubule. Although MAP4 was considered to be long, thin and flexible molecule, never the Tail region may contribute to be the proper folding of MAP4, and/or may interact with other molecules. We concluded that both the Pro-rich region and the AP sequence region take part in the promotion of tubulin polymerization, and that the former is important for the lateral protofilament-protofilament interaction, and the latter is important for the longitudinal affinity between each tubulin dimer in a protofilament.

Myelodysplastic syndrome resembling chronic myeloproliferative disorders in clinicopathological aspects.

We report on a patient with myelodysplastic syndrome (MDS) which resembled chronic myeloproliferative disorder (CMPD). A 67-year-old male was admitted to our hospital in June 1990. A diagnosis of refractory anemia with excess of blasts in transformation (RAEB-T) was based on the peripheral blood and bone marrow findings on admission. However, since thrombocytosis and bone marrow fibrosis was noted, the patient was diagnosed as MDS with myelofibrosis. Low-dose cytosine arabinoside therapy was performed. Although complete remission could not be achieved, a high quality of life could be maintained by appropriate transfusion. In January 1993, the patient was readmitted because of a marked increase in mature neutrophils, showing a moderate increase of blasts, chromosomal aberration (46,XY,12p-) and hepatosplenomegaly; but no fibrosis of bone marrow was observed. These findings suggested that neoplastic proliferation at the level more differentiated to granulocytic lineage occurred at the terminal stage and that his clinical feature was located between MDS and CMPD. This case may be important in considering various aspects of MDS.

PKA and MPF-activated polo-like kinase regulate anaphase-promoting complex activity and mitosis progression.

Ubiquitin-mediated proteolysis is the key to cell cycle control. Anaphase-promoting complex/cyclosome (APC) is a ubiquitin ligase that targets cyclin B and factors regulating sister chromatid separation for proteolysis by the proteasome and, consequently, regulates metaphase-anaphase transition and exit from mitosis. Here we report that Cdc2-cyclin B-activated Polo-like kinase (Plk) specifically phosphorylates at least three components of APC and activates APC to ubiquitinate cyclin B in the in vitro-reconstituted system. Conversely, protein kinase A (PKA) phosphorylates two subunits of APC but suppresses APC activity. PKA is superior to Plk in its regulation of APC, and Plk activity peaks whereas PKA activity is falling at metaphase. These results indicate that Plk and PKA regulate mitosis progression by controlling APC activity.

Human natural resistance-associated macrophage protein is a new type of microtubule-associated protein.

Natural resistance-associated macrophage protein 1 (NRAMP1) is a putative membrane protein that dominates natural resistance to infection. An NRAMP1-glutathione S-transferase fusion protein was used to test the ability of the NRAMP1 NH2-terminal domain to bind to taxol-stabilized microtubules. Co-sedimentation analysis showed that the fusion protein binds to microtubules. Although the NH2-terminal domain of the NRAMP1 molecule has structural homology with the Pro-rich region of microtubule-associated protein 4 (MAP4), the presence of the MAP4 microtubule-binding domain fragment had little effect on the binding of the fusion protein to microtubules.

Evidence for structural differences between the two highly homologous actin-regulatory proteins, destrin and cofilin.

The amino acid sequences of destrin and cofilin are very similar (84% homology) throughout the entire range of proteins, but they have different functions. In this study, we constructed a new cofilin expression plasmid, which had high expression frequency, and the structures of destrin and cofilin were analyzed by limited proteolysis and circular dichroism (CD). When destrin was digested by trypsin, two fragments of 17.0 kDa and 9.2 kDa were obtained, whereas only one 8.4 kDa fragment was obtained from cofilin. In spite of the overall sequence homology, an N-terminal amino acid sequence analyses of the fragments revealed the cleavage sites on destrin and cofilin to be different. These results suggest that destrin and cofilin differ in their overall tertiary folds. Cofilin showed activity similar to destrin at high pH values, although no pH-dependent structural change in cofilin was confirmed by using limited proteolysis and CD.