IgCAMs: bidirectional signals underlying neurite growth.

Understanding how immunoglobulin superfamily cell adhesion molecules (IgCAMs) regulate nervous system development has lagged behind studies on integrins and cadherins. The recent characterization of IgCAM structures combined with cell biological studies on protein-protein interactions and membrane targeting/trafficking demonstrate that IgCAMs interact in exceedingly complex ways to regulate axonal growth and pathfinding.

Structural and functional analysis of the apoptosis-associated tyrosine kinase (AATYK) family.

Apoptosis-associated tyrosine kinase (AATYK) is a protein kinase that is predominantly expressed in the nervous system and is involved in apoptosis and neurite growth of cerebellar granule cells. In this study, we cloned three new members of the mouse AATYK family, AATYK1B, AATYK2 and AATYK3. AATYK1B is a splicing variant of the previously reported AATYK1 (referred to as AATYK1A hereafter). In comparison with AATYK1A, these three AATYK members were characterized by having an extra N-terminal region that consists of a signal peptide-like sequence and a predicted transmembrane (TM) region, which is followed by a kinase domain and a long C-terminal domain. Both TM-containing AATYK isoforms (AATYK(+)TM: AATYK1B, 2, and 3) and TM-lacking isoform (AATYK(-)TM: AATYK1A) were recovered in membrane fractions, suggesting that AATYK(+)TM and AATYK(-)TM are transmembrane- and peripheral-membrane protein kinases, respectively. AATYK1A was recovered in the soluble fraction when the cells were treated with 2-bromo palmitate, suggesting that AATYK1A associates with membrane via palmitoylation. The kinase domain was highly conserved among all AATYK members and was shown to be catalytically active. Three AATYK family members were predominantly expressed in adult mouse brains with almost similar expression profiles: widespread distribution over the various brain regions, especially in the cerebellum and hippocampus, and up-regulated expression during development of the cerebellum. In cultured cerebellar granule cells, AATYK1 was abundantly localized in both soma and axons, AATYK2 distribution was restricted to soma, and AATYK3 was punctately present over the cells. AATYK1 was concentrated in the central domain of growth cones of dorsal root ganglion neurons. Our results indicate that AATYK family members are brain-dominant and membrane-associated kinases with slightly different distribution patterns in the developing and adult mouse brain, which may be involved in fine regulation of neuronal functions including neurite extension and apoptosis.

Optogenetic activation of axon guidance receptors controls direction of neurite outgrowth.

Growth cones of extending axons navigate to correct targets by sensing a guidance cue gradient via membrane protein receptors. Although most signaling mechanisms have been clarified using an in vitro approach, it is still difficult to investigate the growth cone behavior in complicated extracellular environment of living animals due to the lack of tools. We develop a system for the light-dependent activation of a guidance receptor, Deleted in Colorectal Cancer (DCC), using Arabidopsis thaliana Cryptochrome 2, which oligomerizes upon blue-light absorption. Blue-light illumination transiently activates DCC via its oligomerization, which initiates downstream signaling in the illuminated subcellular region. The extending axons are attracted by illumination in cultured chick dorsal root ganglion neurons. Moreover, light-mediated navigation of the growth cones is achieved in living Caenorhabditis elegans. The photo-manipulation system is applicable to investigate the relationship between the growth cone behavior and its surrounding environment in living tissue.

Recycling of the cell adhesion molecule L1 in axonal growth cones.

The cell adhesion molecule (CAM) L1 plays crucial roles in axon growth in vitro and in the formation of major axonal tracts in vivo. It is generally thought that CAMs link extracellular immobile ligands with retrogradely moving actin filaments to transmit force that pulls the growth cone forward. However, relatively little is known about the fate of CAMs that have been translocated into the central (C)-domain of the growth cone. We have shown previously that L1 is preferentially endocytosed at the C-domain. In the present study, we further analyze the subcellular distribution of endocytic organelles containing L1 at different time points and demonstrate that internalized L1 is transported into the peripheral (P)-domain of growth cones advancing via an L1-dependent mechanism. Internalized L1 is found in vesicles positioned along microtubules, and the centrifugal transport of these L1-containing vesicles is dependent on dynamic microtubules in the P-domain. Furthermore, we show that endocytosed L1 is reinserted into the plasma membrane at the leading edge of the P-domain. Monitoring recycled L1 reveals that it moves retrogradely on the cell surface into the C-domain. In contrast, the growth cone advancing independently of L1 internalizes and recycles L1 within the C-domain. For the growth cone to advance, the leading edge needs to establish strong adhesive interactions with the substrate while attachments at the rear are released. Recycling L1 from the C-domain to the leading edge provides an effective way to create asymmetric L1-mediated adhesion and therefore would be critical for L1-based growth cone motility.

The role of endocytic l1 trafficking in polarized adhesion and migration of nerve growth cones.

Motility of the nerve growth cone is highly dependent on its dynamic interactions with the microenvironment mediated by cell adhesion molecules (CAMs). These adhesive interactions can be spatially regulated by changing the density and avidity of CAMs on the growth cone. Previous studies have shown that L1, a member of the immunoglobulin superfamily of CAMs, is endocytosed at the central domain of the growth cone followed by centrifugal vesicular transport and reinsertion into the plasma membrane of the leading edge. The present paper focuses on the functional significance of endocytic L1 trafficking in dorsal root ganglia neurons in vitro. We demonstrate that the rate of L1-based neurite growth has a positive correlation with the amount of endocytosed L1 in the growth cone, whereas stimulation of neurite growth via an N-cadherin-dependent mechanism does not increase L1 endocytosis. A growth cone that migrates on an L1 substrate exhibits a steep gradient of L1-mediated adhesion (strong adhesion at the growth cone's leading edge and weak adhesion at the central domain). This gradient of L1 adhesion is attenuated after inhibition of L1 endocytosis in the growth cone by intracellular loading of a function-blocking antibody against alpha-adaptin, a subunit of the clathrin-associated AP-2 adaptor. Inhibition of L1 endocytosis by this antibody also decreased the rate of L1-dependent growth cone migration. These results indicate that the growth cone actively translocates CAMs to create spatial asymmetry in adhesive interactions with its environment and that this spatial asymmetry is important for growth cone migration.

Synergistic inhibition of human gastric carcinoma cell growth by 1-beta-D-arabinofuranosylcytosine and hydroxyurea or 2'-deoxyguanosine in vitro.

The cytotoxicity of 1-beta-D-arabinofuranosylcytosine (ara-C) in combination with hydroxyurea (HU) or 2'-deoxyguanosine (GdR) on human gastric carcinoma MK-1 cells and colon carcinoma HT-15 cells was studied. Synergistic interaction between ara-C and HU on MK-1 cells and HT-15 cells, or ara-C and GdR on MK-1 cells was shown using the combination index method. HU increased the accumulation of ara-C triphosphate (ara-CTP) in the acid-soluble pool and diminished the cellular deoxyCTP (dCTP) pool. HU had no effect on the incorporation of ara-C into DNA and RNA. These results indicate that HU-induced elevation in ara-CTP and decrease in dCTP are the basis for synergy among ara-C and HU in MK-1 cells. GdR diminished cellular dCTP slightly, but it decreased the accumulation of ara-CTP in the acid-soluble pool and did not increase the incorporation of ara-C into DNA. On the other hand, ara-C increased cellular deoxyGTP (dGTP) level in the presence of GdR. These results indicate that synergy between ara-C and GdR is mediated through increased cellular dGTP which might inhibit DNA synthesis directly.

Interleukin-1ß and tumor necrosis factor-α co-operatively enhance a novel trophic activity of astrocytes for pontine cholinergic neurons in vitro.

Insulin and insulin-like growth factors (IGFs) are the only known trophic factors for pontine cholinergic neurons. The present study revealed that astrocyte-extract pretreated with IL-1ß and TNF-α significantly enhanced choline acetyltransferase (ChAT) activity of the pontine neurons in the presence of a supramaximal dose of insulin, while various trophic factors including IGFs failed to increase the ChAT activity under the same culture conditions, suggesting that IL-1ß and TNF-α co-operatively enhanced the expression of a novel trophic factor for pontine cholinergic neurons in astrocytes.

Accidentally detected brain tumors: clinical analysis of a series of 110 patients.

The clinical records of 1,155 patients with 1,159 brain tumors who drained on Keio University Hospital between 1983 and 1994 were reviewed. Apparently asymptomatic patients and those whose complaints or neurological deficits were not caused by the brain tumors were defined as accidental cases. For example, patients with a headache which was considered to be unrelated to the presence of a tumor were included in this series. One hundred and ten (9.5%) of the 1,155 cases were found to be accidental. Since three accidental cases had multiple meningiomas, there were 113 accidental brain tumors which involved 63 meningiomas, 22 pituitary adenomas, 9 gliomas, 7 metastatic carcinomas. 5 acoustic neurinomas and 7 miscellaneous. Meningiomas occurred significantly more frequent than other types of accidentally identified tumors. Convexity meningiomas and falx meningiomas accounted for 53.9% of the accidental meningiomas, whereas parasagittal meningiomas were less frequent. It is of note that three out of four cases with multiple meningiomas were accidental. Comparison between the present results and the previously reported incidence of asymptomatic brain tumors in postmortem studies suggest that a substantial number of pituitary adenomas, acoustic neurinomas and small parasagittal meningiomas without suggestive symptoms are likely to be missed by routine neuroradiological examinations.

Cholesterol granuloma of the petrous apex: establishment of a drainage route into the superior tympanic cavity--technical note.

A 40-year-old male presented with a cholesterol granuloma of the petrous apex manifesting as progressive hearing loss and tinnitus. The lesion was treated via an extradural middle cranial fossa approach employing a new procedure to establish a drainage pathway into the superior tympanic cavity which preserved his hearing. The pathway was formed by a groove 5 mm wide and deep in the anterolateral aspect of the petrous bone, crossing the major petrosal nerve and carotid artery, running around the cochlea, crossing the tensor tympanic muscle, and entering the superior tympanic cavity above the orifice of the eustachian tube. This procedure is easy to perform without special techniques.

Accumulation of nerve growth factor in cerebrospinal fluid and biological activity following neurosurgery.

Perioperative nerve growth factor (NGF) levels in cerebrospinal fluid (CSF) of patients with acoustic neurinoma (14 cases), tentorial meningioma (1 case), or subarachnoid hemorrhage (1 case) were examined. Preoperative NGF levels in CSF were below the level of detection in all patients. However, NGF was found to accumulate transiently in CSF following neurosurgery. Pre- and postoperative CSF obtained from a patient with acoustic neurinoma enhanced the proliferation of astrocytes in neuronal cell cultures derived from embryonic rat cortex grown in serum-free defined medium, and increased choline acetyltransferase activity of cholinergic neurons derived from embryonic rat septal area and brainstem. The effect of postoperative CSF on septal and brainstem neurons was more potent than that of preoperative CSF. These results indicate that NGF and non-NGF-type neurotrophic activities accumulate in the CSF following neurosurgery. These neurotrophic activities are probably important in the regeneration of damaged neural networks in the central nervous system.

Unusual location of intracranial vagus neurinoma--case report.

A 55-year-old male presented with hearing disturbance and tinnitus in the left ear. Computed tomography (CT) and magnetic resonance imaging demonstrated a well-defined, homogeneously enhanced mass in the left cerebellomedullary cistern without extension close to the jugular foramen. A three-dimensional image reconstructed from thin-slice CT scans demonstrated that the mass was clearly separated from the jugular foramen. The mass lesion was totally removed surgically. At surgery the tumor was found to originate from one rootlet of the vagal nerve just after its exit from the medulla oblongata. The histological diagnosis was neurinoma. Intracranial neurinomas of the glossopharyngeal, vagal, or accessory nerve usually originate within or close to the jugular foramen. This unusual location made it difficult to achieve a correct preoperative diagnosis.

Objective monitoring of graft-versus-host disease: alpha 1-antichymotrypsin concentration changes after bone marrow transplantation in patients developing graft-versus-host reactions.

The levels of alpha 1-antichymotrypsin (ACT) was monitored in patients who underwent bone marrow transplantation. Seven received HLA-identical sibling bone marrow grafts, two received transplants from twins and one was given HLA-nonidentical marrow from his father. A dramatic increase of ACT was observed in all patients who developed graft-versus-host disease (GvHD). ACT did not rise at all in the case of patients who received marrow from twins, even in a patient who was given three transplants from the same donor. The patient transplanted from his father died from GvHD and the increase of ACT was the greatest fluctuation measured.

The isoelectric focusing pattern of desialylated alpha 1-antichymotrypsin of heterozygous alpha 1-antichymotrypsin deficiency and of acute phase plasma.

In an attempt to find a molecular marker for the putative abnormal allele in heterozygous alpha 1-antichymotrypsin (ACT) deficiency (a rare trait associated with early emphysema, childhood asthma and chronic "cryptogenic" liver disease) the isoelectric focusing pattern of neuraminidase treated plasma samples from subjects of ACT deficiency families as well as acute phase plasma were compared. There was no difference in the isoform pattern of plasma from ACT deficiency heterozygotes, normal subjects or patients with acute phase response. However, in acute phase plasma there was a disproportional increase in two isoforms, one of which conceivably may be used to mark the early phase of the acute phase response.

Isolation, purification and identification of a serum DNA binding protein which increases in malignant diseases.

A DNA binding protein in human serum was purified and identified. This protein was isolated from pooled human serum by DEAE Sephadex column chromatography, DNA cellulose affinity column chromatography, Sephadex G-200 gel filtration and Sepharose 6B gel filtration. It was found that the purified protein consisted of two subunits, one with a molecular weight of 80,000 and the other 25,000, evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The purified protein was in fact IgM determined by physicochemical and immunochemical characteristics.

Purification, properties and identification of a serum DNA binding protein (64DP) and its microheterogeneity.

A DNA binding protein with a molecular weight of 64,000, designated 64DP, has been purified and characterized. This protein was isolated from adult human pooled serum by DEAE Sephadex column Chromatography, DNA cellulose affinity column chromatography, ammonium sulfate fractionation and Sephadex G-150 gel filtration. The final preparation of 64DP was homogeneous, as judged from polyacrylamide gel electrophoresis with or without sodium dodecyl sulfate and sedimentation experiments. Physicochemical and immunochemical properties of this protein were very similar or identical to those of alpha-1-antichymotrypsin with some differences in electric mobility and th pattern of isoelectric focusing. Furthermore, the general properties of 64DP from various sera were practically similar with the exception that isolectric focusing analysis showed microheterogeneity among 64DP purified from various sera.

Neural cell adhesion molecule L1: signaling pathways and growth cone motility.

The neural cell adhesion molecule L1 plays a key role in nervous system development including neuronal migration, neurite growth, and axonal fasciculation. L1 is expressed on most developing axons, and homophilic binding of L1 molecules on adjacent axons is likely to play a key role in axon extension. It is now well documented that a number of second-messenger systems are involved in L1-stimulated neurite growth in vitro. However, it is unclear how L1 homophilic or heterophilic binding trigger signals that regulate the mechanical forces that produce axon extension. In this report, we will review recent advances in understanding L1-associated signals, L1 interactions with the cytoskeleton, and the molecular mechanisms underlying growth cone motility.