Activation of ATPase Activity in the Chromatin Fraction of Pea Nuclei by Calcium and Calmodulin

ATPase, especially the Ca^2$-dependent enzyme, in the chromatinfraction isolated from nuclei of pea seedlings was activatedby bovine brain calmodulin and by protein that was judged tobe calmodulin from its Ca^2$-dependent activation of NAD kinase.This calmodulin-dependent ATPase activity was blocked by calmodulin-specificinhibitors. (Received July 11, 1983; Accepted October 24, 1983)     

Changes in predominant energy substrate after hepatectomy

The changes in the energy substrate utilized by the remnant liver were studied in relation to the changes in the cellular energy status of 25 and 70% hepatectomized rabbits. In 25% hepatectomized rabbits, the energy charge $\text{(}\text{(}\text{ATP}\text{+0.5}\text{ADP}\text{)}\text{(}\text{ATP}\text{+}\text{ADP}\text{+}\text{AMP}\text{)}\text{)}$ level of the remnant liver remained unchanged, the energy substrate of which was predominantly glucose, rather than fatty acid. In contrast, in 70% hepatectomized rabbits, the energy production by the mitochondria was mainly dependent upon fatty acid oxidation at the early period after hepatectomy when the energy charge level decreased remarkably, and then upon glucose oxidation, concomitant with the restoration of the energy charge. It is suggested that the changes in the energy substrate utilized are closely related to those in the energy charge level and the mitochondrial phosphorylative activity of the remnant liver following hepatectomy.     

Molecular cloning and characterization of a novel human STE20-like kinase, hSLK

We have cloned a human counterpart to a guinea pig STE20-like kinase cDNA, designated human SLK (hSLK), from a human lung carcinomatous cell line A549 cDNA library. hSLK cDNA encodes a novel 1204 amino acid serine/threonine kinase for which the kinase domain located at the N-terminus shares considerable homology to that of the STE20-like kinase family. The C-terminal domain of hSLK includes both the coiled-coil structure and four Pro/Glu/Ser/Thr-rich (PEST) sequences, but not the GTPase-binding domain (GBD) that is characteristic of the p21-activated kinase (PAK) family, polyproline consensus binding sites, or the Leu-rich domain seen in the group I germinal center kinases (GCKs). Northern blot analysis indicated that hSLK was ubiquitously expressed. hSLK overexpressed in COS-7 cells phosphorylates itself as well as myelin basic protein used as a substrate. On the other hand, hSLK cannot activate any of the three well-characterized mitogen-activated protein kinase MAPK (ERK, JNK/SAPK and p38) pathways. Moreover, hSLK kinase activity is not upregulated by constitutive active forms of GTPases (RasV12, RacV12 and Cdc42V12). These structural and functional properties indicate that hSLK should be considered to be a new member of group II GCKs.     

Chronic Alterations in Monoaminergic Cells in the Locus Coeruleus in Orexin Neuron-Ablated Narcoleptic Mice

Narcolepsy patients often suffer from insomnia in addition to excessive daytime sleepiness. Narcoleptic animals also show behavioral instability characterized by frequent transitions between all vigilance states, exhibiting very short bouts of NREM sleep as well as wakefulness. The instability of wakefulness states in narcolepsy is thought to be due to deficiency of orexins, neuropeptides produced in the lateral hypothalamic neurons, which play a highly important role in maintaining wakefulness. However, the mechanism responsible for sleep instability in this disorder remains to be elucidated. Because firing of orexin neurons ceases during sleep in healthy animals, deficiency of orexins does not explain the abnormality of sleep. We hypothesized that chronic compensatory changes in the neurophysiologica activity of the locus coeruleus (LC) and dorsal raphe (DR) nucleus in response to the progressive loss of endogenous orexin tone underlie the pathological regulation of sleep/wake states. To evaluate this hypothesis, we examined firing patterns of serotonergic (5-HT) neurons and noradrenergic (NA) neurons in the brain stem, two important neuronal populations in the regulation of sleep/wakefulness states. We recorded single-unit activities of 5-HT neurons and NA neurons in the DR nucleus and LC of orexin neuron-ablated narcoleptic mice. We found that while the firing pattern of 5-HT neurons in narcoleptic mice was similar to that in wildtype mice, that of NA neurons was significantly different from that in wildtype mice. In narcoleptic mice, NA neurons showed a higher firing frequency during both wakefulness and NREM sleep as compared with wildtype mice. In vitro patch-clamp study of NA neurons of narcoleptic mice suggested a functional decrease of GABAergic input to these neurons. These alterations might play roles in the sleep abnormality in narcolepsy.     

Metamorphopsia Associated with Branch Retinal Vein Occlusion

PurposeTo apply M-CHARTS for quantitative measurements of metamorphopsia in eyes with acute branch retinal vein occlusion (BRVO) and to elucidate the pathomorphology that causes metamorphopsia.MethodsThis prospective study consisted of 42 consecutive patients (42 eyes) with acute BRVO. Both at baseline and one month after treatment with ranibizumab, metamorphopsia was measured with M-CHARTS, and the retinal morphological changes were examined with optical coherence tomography.ResultsAt baseline, metamorphopsia was detected in the vertical and/or horizontal directions in 29 (69.0%) eyes; the mean vertical and horizontal scores were 0.59 ± 0.57 and 0.52 ± 0.67, respectively. The maximum inner retinal thickness showed no association with the M-CHARTS score, but the M-CHARTS score was correlated with the total foveal thickness (r = 0.43, p = 0.004), the height of serous retinal detachment (r = 0.31, p = 0.047), and the maximum outer retinal thickness (r = 0.36, p = 0.020). One month after treatment, both the inner and outer retinal thickness substantially decreased. However, metamorphopsia persisted in 26 (89.7%) of 29 eyes. The posttreatment M-CHARTS score was not correlated with any posttreatment morphological parameters. However, the posttreatment M-CHARTS score was weakly correlated with the baseline total foveal thickness (r = 0.35. p = 0.024) and closely correlated with the baseline M-CHARTS score (r = 0.78, p < 0.001).ConclusionsMetamorphopsia associated with acute BRVO was quantified using M-CHARTS. Initial microstructural changes in the outer retina from acute BRVO may primarily account for the metamorphopsia.     

Determination of muscimol and ibotenic acid in Amanita mushrooms by high-performance liquid chromatography and liquid chromatography-tandem mass spectrometry

A reliable analytical method was developed for the quantification and identification of muscimol (MUS) and ibotenic acid (IBO), the toxic constituents of Amanita muscaria and Amanita pantherina. MUS and IBO were extracted from mushrooms by aqueous methanol and derivatized with dansyl chloride (DNS-Cl). After extraction with ethyl acetate and evaporation of the solvent, the residue was ethylated with 1.25 M hydrogen chloride in ethanol. The resulting derivatives were quantified by high-performance liquid chromatography with UV detection and identified by liquid chromatography electrospray ionization tandem mass spectrometry. Calibration curves were linear in the range of 25-2500 ppm for MUS and 40-2500 ppm for IBO, respectively. This method was successfully applied to identify and quantify MUS and IBO in Amanita mushrooms naturally grown and circulated in the drug market.     

Intraflagellar transport genes are essential for differentiation and survival of vertebrate sensory neurons

Cilia play diverse roles in vertebrate and invertebrate sensory neurons. We show that a mutation of the zebrafish oval (ovl) locus affects a component of the ciliary transport (IFT) mechanism, the IFT88 polypeptide. In mutant retina, cilia are generated but not maintained, producing the absence of photoreceptor outer segments. A loss of cilia also occurs in auditory hair cells and olfactory sensory neurons. In all three sense organs, cilia defects are followed by degeneration of sensory cells. Similar phenotypes are induced by the absence of the IFT complex B polypeptides, ift52 and ift57, but not by the loss of complex A protein, ift140. The degeneration of mutant photoreceptor cells is caused, at least partially, by the ectopic accumulation of opsins. These studies reveal an essential role for IFT genes in vertebrate sensory neurons and implicate the molecular components of intraflagellar transport in degenerative disorders of these cells.     

Regulation of Lck and Fyn tyrosine kinase activities by transmembrane protein tyrosine phosphatase leukocyte common antigen-related molecule

Leukocyte common antigen-related molecule (LAR) is a receptor-like protein tyrosine phosphatase (PTPase) with two PTPase domains. In the present study, we detected the expression of LAR in the brain, kidney, and thymus of mice using anti-LAR PTPase domain subunit monoclonal antibody (mAb) YU1. In the thymus, LAR was expressed on CD4(-)CD8(-) and CD4(-)CD8(low) thymocytes. The development of thymocytes in CD45 knockout mice is blocked partially in the maturation of CD4(-)CD8(-) to CD4(+)CD8(+). We postulated that LAR regulates Lck and Fyn in the immature thymocytes. Transfection of wild-type LAR activated extracellular signal-regulated kinase signal transduction pathway in CD45-deficient Jurkat cells stimulated with anti-CD3 mAb. LAR mutants, with Cys to Ser mutation in the catalytic center of PTPase D1, bound to tyrosine-phosphorylated Lck and Fyn, and LAR PTPase domain 2 was tyrosine phosphorylated by Fyn tyrosine kinase. The phosphorylated LAR was associated with Fyn Src homology 2 domain. Moreover, LAR dephosphorylated phosphorylated tyrosine residues in both the COOH terminus and kinase domain of Fyn in vitro. Our results indicate that Lck and Fyn would be substrates of LAR in immature thymocytes and that each LAR PTPase domain plays distinct functional roles in phosphorylation and dephosphorylation.