Molecular mechanisms in synaptic vesicle endocytosis.
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Animals↗
Biomedical subjects
K Takei
Publications and source records attributed to K Takei.
[Clinical assessment for prognostic factors related to recurrence and progression in superficial bladder cancer].
PURPOSE: The objective of this assessment is to identify prognostic factors of recurrence and disease progression of primary superficial transitional cell carcinoma of the bladder. PATIENTS AND METHODS: TUR-Bt was performed in 150 patients with initial superficial bladder cancer, of which pathological diagnosis was pTa or pT1 transitional cell carcinoma. The recurrence and progression free survival was examined. The clinicopathological factors analyzed were as follows: grade, pT, tumor size, tumor number and tumor form. RESULTS: Median follow-up period was 39 months (range 3-184). The age distribution was from 25 to 98 years old with the average of 67. The patients were 122 males and 28 females. Recurrence was observed in 72 patients. The 5-year recurrence free rate for all cases were 41.6%. The factors found to be of significance for the prognosis for recurrence were tumor size (1 cm <) and tumor multiplicity (p < 0.01). Progression was seen in 15 patients; 8 invasive tumors, 2 lymph node metastases and 5 distant metastases. Twelve cases of those died of cancer. The 5-year progression free rate for all cases were 87.0%. The prognostic factors related to progression found in this study were G3, pT1 and sessile form (p < 0.01). There was no significant difference in recurrence free rate between cases with and without postoperative therapy; UFT p.o., ADM i.i. (intravesical instillation), ADM + Ara-C i.i. and PEP + 5-FU + Ara-C i.i. CONCLUSION: Tumor size more than 1 cm and tumor multiplicity were the high risk factors about recurrence. So were G3, pT1 and sessile form about progression.
Adult↗
Molecular subtyping of clinical isolates of Escherichia coli from patients with lower urinary tract infections in Japan.
Crossed-field gel electrophoresis was used to analyse the chromosomal genomic DNA from 41 clinical isolates of Escherichia coli serotypes O-2, O-4, O-6 and O-75 from patients with lower urinary tract infections after digestion of the DNA with Not I and Xba I, respectively. Not I generated 28 distinctly different genomic profiles after crossed-field gel electrophoresis (CFGE) from 34 strains and Xba I generated 35 distinctly different genomic profiles from 41 strains. Not I failed to cleave the genomic DNA from one strain of E. coli serotype O-6 and from all of six strains of serotype O-75. Both enzymes were shown to have equal potential utility for detection of variability in CFGE profiles among 34 strains because both classified 34 of the 41 strains into 28 profiles in a similar manner. No examples of essentially identical restriction profiles among strains of different serotypes were obtained.
Bacterial Typing Techniques↗
[The effect of subconjunctival injection of endothelin-1 on intraocular pressure in the rabbit].
Endothelin-1 (ET-1) is a potent vasoconstrictive peptide produced in part by vascular endothelial cells. In order to investigate its effects on the episcleral vascular system and intraocular pressure (IOP), we injected ET-1 (3, 10, 30, 100, 300, 1,000 pmol) into the subconjunctival space of rabbits and measured IOP with a manometer. Injection of a dose higher than 10 pmol caused a transient increase of IOP. Mean maximum elevation rate of IOP for each dose of ET-1 was 14.9 +/- 0.9%, (mean +/- standard error) 43.5% +/- 9.5%, 40.8 +/- 7.5%, 46.9 +/- 9.8%, and 84.1 +/- 22.6. Next, we injected 1,000 pmol into the subconjunctival space, and continuously measured IOP and ocular pulse pressure with a manometer. IOP increased rapidly after ET-1 injection. Maximal increase of IOP was observed at 22.7 +/- 9.2 min after ET-1 injection, and IOP decreased after the peak. The ocular pulse pressure increased with IOP elevation and decreased with the IOP reduction. We speculated that the transient elevation of IOP was caused by increase of aqueous outflow resistance, and the decrease of IOP was caused by decrease of aqueous outflow resistance and decrease of blood flow in the ciliary body and the choroid. This strongly suggests that subconjunctival injection of ET-1 could have a large effect on the episcleral vascular system, aqueous outflow, and blood flow in the ciliary body and the choroid.
Animals↗
[A case of leiomyosarcoma of the diverticulum of urinary bladder].
A 77-year-old man was admitted to our hospital complaining of gross hematuria. Cystoscopy showed an approximately 4-cm non-papillary tumor in and out of the diverticulum of the left posterior wall. Total cystectomy was performed. Histopathological diagnosis was pleomorphic leiomyosarcoma. According to TNM classification of bladder cancer, the stage of this tumor was pT3bpN0M0. The patient had local recurrence two months after the operation, and died a month later. This is the second case of leiomyosarcoma of the diverticulum of urinary bladder reported in Japan.
Aged↗
Optimum form of posterior chamber intraocular lenses to minimize aberrational astigmatism.
To optically determine the optimum form for a posterior chamber intraocular lens (PC IOL), we calculated the aberrational astigmatism induced by tilt and decentration of the PCIOL using an exact raytracing. First, the position and the radii of curvatures of the IOL were determined to make an emmetropic eye model using a paraxial raytracing. Next, the chief rays originating from the fovea centralis were traced backward through the tilted and/or decentrated PC IOL, the center of the pupil and the cornea, using trigonometric raytracing. Finally, the maximum and minimum aberrational astigmatism were calculated based on the Coddington's Equations for the sagittal and the tangential foci of the ray. All the refractive parameters in Gullstrand's No. 1 schematic eye were adopted. The effect of varying anterior corneal asphericity on the results was also examined. Four forms of polymethylmethacrylate PC IOLs (refractive index: 1.491) were analyzed; a plano-convex IOL with the curved surface facing the cornea, and three bi-convex forms with the ratio of anterior-to-posterior radii of curvatures of 1:4, 1:2 and 1:1, respectively. The 1:4 bi-convex form showed the lowest values for the maximum aberrational astigmatism calculated at every combination of tilt and decentration except 0 degrees tilt and/or 0 mm decentration. The aberrational astigmatism with the 1:4 bi-convex form of PC IOL did not exceed 1.0 D at the maximum tilt and decentration. The variation of anterior corneal asphericity did not influence the results. We conclude that the 1:4 bi-convex form of PC IOL minimizes the postoperative astigmatism induced by tilt and/or decentration of the lens.
Astigmatism↗
p145, a major Grb2-binding protein in brain, is co-localized with dynamin in nerve terminals where it undergoes activity-dependent dephosphorylation.
Three major rat brain proteins were recently found to bind the SH3 domains of Grb2: synapsin I, dynamin, and a novel 145-kDa protein (p145) (McPherson, P. S., Czernik, A. J., Chilcote, T. J., Onofri, F., Benfenati, F., Greengard, P., Schlessinger, J., and De Camilli, P. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 6486-6490). We have now used antibodies raised against p145 which had been purified by Grb2 affinity chromatography and SDS-polyacrylamide gel electrophoresis to characterize this protein. p145 is neuron-specific and co-distributes with dynamin in subcellular fractions of rat brain. Both proteins are partially recovered in soluble and particulate fractions. By immunofluorescence, p145 is closely co-localized with dynamin, which we show here to be highly concentrated in nerve terminals. In contrast to synapsin I, which is highly enriched in a purified synaptic vesicle fraction, both p145 and dynamin are de-enriched in this fraction. However, both proteins are found on membranes which are immunoisolated with antibodies directed against the synaptic vesicle membrane protein synaptophysin, suggesting that dynamin and p145 are localized in part on organelles which represent intermediate stages in the reformation of synapatic vesicles during recycling. Finally, we have determined that p145, like dynamin, is a phosphoprotein which undergoes dephosphorylation in response to nerve terminal depolarization. These findings suggest that p145 participates with dynamin in synaptic vesicle endocytosis and recycling.
Adaptor Proteins, Signal Transducing↗
Synaptic targeting domains of synapsin I revealed by transgenic expression in photoreceptor cells.
Synapsins are abundant nerve terminal proteins present at all synapses except for ribbon synapses, e.g. photoreceptor cell synapses. Multiple functions have been proposed for synapsins, including clustering of synaptic vesicles and regulation of synaptic vesicle exocytosis. To investigate the physiological functions of synapsin and to ascertain which domains of synapsin are involved in synaptic targeting in vivo, we expressed synapsin Ib and its N- and C-terminal domains in the photoreceptor cells of transgenic mice. In these cells synapsin Ib is targeted efficiently to synaptic vesicles but has no significant effect on the development, structure or physiology of the synapses. This suggests that synapsin I does not have dominant physiological or morphoregulatory functions at these synapses. Full-length synapsin Ib and the N-terminal domains of synapsin Ib but not its C-terminal domains are transported to synapses, revealing that the molecular apparatus for synaptic targeting of synapsins is also present in cells which form ribbon synapses that normally lack synapsins. This apparatus appears to utilize the conserved N-terminal domains that are shared between all synapsins.
Animals↗
The role of Rab3A in neurotransmitter release.
The small GTP-binding protein Rab3A is a Rab family member that is abundant in brain synaptic vesicles. Here we show that mice in which the rab3A gene has been mutated by homologous recombination do not express Rab3A but are viable and fertile. Electrophysiological recordings in hippocampal CA1 pyramidal cells indicate that most of their synaptic parameters are also normal, although synaptic depression after short trains of repetitive stimuli (15-30 stimuli at 14 Hz) is significantly increased. Levels of the Rab3A-binding protein rabphilin are decreased by 70%, but expression of more than 20 other synaptic proteins is unchanged. No compensatory changes were detected in other GTP-binding proteins or in proteins that interact with Rab3. Rab3A thus appears not to be essential for synaptic vesicle exocytosis but to play a role in the recruitment of synaptic vesicles for exocytosis during repetitive stimulation.
Adaptor Proteins, Signal Transducing↗
Synaptic targeting of rabphilin-3A, a synaptic vesicle Ca2+/phospholipid-binding protein, depends on rab3A/3C.
rab3A, a low molecular weight GTP-binding protein of synaptic vesicles with a putative function in synaptic vesicle docking, interacts in a GTP-dependent manner with rabphilin-3A, a peripheral membrane protein that binds Ca2+ and phospholipids. We now show that rabphilin-3A is an evolutionarily conserved synaptic vesicle protein that is attached to synaptic vesicle membranes via its N terminus and exhibits a heterogeneous distribution among synapses. In rab3A-deficient mice, rabphilin-3A is decreased in synapses belonging to neurons that primarily express rab3A and accumulates in the perikarya of these neurons. In contrast, neurons expressing significant levels of rab3C still contain normal levels of rabphilin-3A in a synaptic pattern, and rabphilin-3A binds rab3C in vitro. These results suggest that analogous to the membrane recruitment of raf by ras, rab3A and rab3C may function in recruiting rabphilin-3A to the synaptic vesicle membrane in a GTP-dependent manner.
Adaptor Proteins, Signal Transducing↗
Inositol 1,4,5-trisphosphate receptor causes formation of ER cisternal stacks in transfected fibroblasts and in cerebellar Purkinje cells.
The inositol 1,4,5-trisphosphate receptor (IP3R) is expressed at very high levels in cerebellar Purkinje cells. Within these neurons, it has a widespread distribution throughout the endoplasmic reticulum (ER) and is present at particularly high concentrations at sites of membrane appositions within peculiar stacks of ER cisternae. Here we report that stacks of ER cisternae, reminiscent of those observed in Purkinje cells, can be induced by overexpression of full-length IP3R, but not of mutant forms of the protein in COS cells. Within these stacks the IP3R forms a crystalline array at apposed cisternal faces. Additionally, we show that Purkinje cell stacks are not permanent structures. Our findings suggest that massive stack formation in purkinje cells represents an adaptive response of the ER to hypoxic conditions and is due to the presence of the high concentration of IP3R in its membranes.
Animals↗
Localization of Rab5 to synaptic vesicles identifies endosomal intermediate in synaptic vesicle recycling pathway.
After exocytosis, synaptic vesicles rapidly endocytose and recycle but little is known about the molecular mechanisms involved. Rab5 is a ubiquitous low molecular weight GTP-binding protein required for endosomal fusion in fibroblasts. We have now raised polyclonal and monoclonal antibodies to rat Rab5 and show that in rat brain, Rab5 is a major synaptic vesicle protein. Immunoisolation of vesicular organelles from brain with antibodies to either Rab3A and Rab5 as small GTP-binding proteins or with synaptophysin as general synaptic vesicle marker demonstrates that there are overlapping populations of synaptic vesicles containing either Rab5 or Rab3A or both, suggesting a stage-specific association of these low-molecular weight GTP-binding proteins with synaptic vesicles. Our data provide the first biochemical evidence that synaptic vesicle recycling involves an endosomal intermediate similar to that of the receptor-mediated endocytosis pathway.
Amino Acid Sequence↗
Morphologic and biochemical analysis of the intracellular trafficking of the Alzheimer beta/A4 amyloid precursor protein.
Abnormal metabolic processing of the beta/A4 amyloid precursor protein (APP) has been implicated in the pathogenesis of Alzheimer disease. Several aspects of normal APP processing have been elucidated, but the precise cellular trafficking of APP remains unclear. To investigate APP trafficking pathways further, we have examined the subcellular distribution of APP in rat brain tissue and a variety of cultured cell types, and correlated this distribution with the biochemical processing of APP. In immunofluorescence microscopy of rat brain sections, APP immunoreactivity was concentrated in the Golgi complex and in proximal axon segments. In addition, a lower level of punctate fluorescence was visible throughout the neuropil. By immunoelectron microscopy of rat brain tissue fragments, APP was found associated with Golgi elements and with medium-sized, invaginated vesicles in both axons and dendrites. Prominent localization of APP to the Golgi complex was also found in primary cultures of rat hippocampal neurons and in non-neuronal cell lines. When cultured cells were treated with brefeldin A (BFA), APP immunoreactivity changed from a Golgi-like to an endoplasmic reticulum-like distribution. No APP was detected in the BFA-induced reticulum identified by the transferrin receptor, indicating that concentration of APP in the Golgi does not reflect recycling between the trans-Golgi network and early endosomal system. In immunoblots of BFA-treated cells, there was an accumulation of full-length APP and inhibition of APP secretory processing. Treatment with phorbol ester resulted in a marked elevation of APP secretion, but no obvious redistribution of APP immunoreactivity was apparent at the light microscope level. The lysosomotropic drug chloroquine induced accumulation of APP in cell lysates, as seen by immunoblotting. Immunofluorescence microscopy of chloroquine-treated cells demonstrated a colocalization of APP with the lysosomal marker Igp 120, whereas no colocalization was seen in untreated cells. Taken together, these results support a scheme in which APP is concentrated in the Golgi complex as it travels through the central vacuolar system en route to the plasma membrane for secretion of its amino-terminal domain and/or to lysosomes for degradation.
Alzheimer Disease↗
A new model of transient complete obstruction of retinal vessels induced by endothelin-1 injection into the posterior vitreous body in rabbits.
Endothelin-1 (ET-1) is a potent vasoconstrictor peptide produced by vascular endothelial cells. In order to investigate the effects of ET-1 on retinal vessels, ET-1 (1-1000 pmol) was injected into the posterior vitreous body in rabbits. A high dose of ET-1 induced transient complete obstruction of the retinal vessels. In this experimental model of transient complete obstruction of the retinal vessels, the effects of ET-1 on retinal function were further analyzed by means of electroretinograms. The scotopic a-wave was not affected, but the amplitude of the scotopic b-wave was significantly elevated. The amplitude of oscillatory potentials was significantly reduced. These phenomena suggested that retinal ischemia without choroidal ischemia was brought about due to severe vasoconstriction of the retinal arteries. These findings indicate that intravitreal injection of ET-1 causes a transient cessation of blood supply from retinal vessels and that oscillatory potentials in electroretinograms appear to be sensitive for detecting changes of retinal circulation. This new model of transient complete obstruction of retinal vessels might be useful for studying the pathophysiology of severe retinal ischemia.
Animals↗
Analysis of vasocontractile responses to endothelin-1 in rabbit retinal vessels using an ETA receptor antagonist and an ETB receptor agonist.
To study the role of endothelin receptor subtypes in rabbit retinal arteries, endothelin-1 (ET-1)-induced vasoconstriction was analyzed using the ETA receptor antagonist BQ-123 and the ETB receptor agonist BQ-3020. A cumulative injection of ET-1 (1 approximately 100 pmole) into the posterior vitreous body in anesthetized rabbits caused dose-dependent vasoconstriction in the retinal arteries, and ultimately caused complete obstruction. Fifteen minutes after BQ-123 (1 mumole) was injected into the posterior vitreous body, the dose-response curve of ET-1 was significantly shifted to the right. Intravitreal injection of BQ-3020 caused a significant retinal vasoconstriction only at the highest dose (1000 pmole). Therefore, in rabbit retinal arteries, ET-1-induced vasoconstriction appears to be mediated mainly through ETA receptors.
Animals↗
Major glycoproteins in carp CNS myelin: homology to P0 protein with HNK-1/L2 carbohydrate epitope.
We studied the myelin protein profiles of carp from a phylogenetic point of view. The carp central nerve myelin contained two reactive bands, 28 and 25 kDa, demonstrated with anti-bovine P0 antibody. Their molecular weights are slightly different from those of two positive bands found in carp peripheral myelin. The N-terminal amino acid sequences of these four positive bands were identical to one another and showed high homology with those of mammalian P0 protein, suggesting that carp central myelin contains the P0-like protein. Lectin binding analysis revealed that carbohydrate structure of the P0-like proteins in carp central myelin is similar to those in peripheral myelin of carp and other vertebrates. Further, the carp P0-like glycoproteins, like the P0 proteins of other vertebrates, reacted with antibodies that recognize the HNK-1/L2 carbohydrate epitope. We conclude that the major structural glycoprotein in central myelin of the carp is homologous to P0 protein in peripheral myelin of other higher classes of vertebrates.
Amino Acid Sequence↗
Expression of a P0-like glycoprotein in central nervous system myelin of amphibians (Ambystoma mexicanus, Xenopus laevis and Rana catesbeiana).
1. The myelin protein profiles in the CNS and PNS of three species of amphibians were analyzed by biochemical and immunohistochemical methods. 2. The CNS myelin of the African clawed frog (Xenopus) and the Mexican salamander (axolotl) contained, in addition to proteolipid protein, a unique protein zero (P0)-like protein, whereas the adult bullfrog did not. 3. A strong expression of the P0-like protein in the bullfrog CNS myelin was found transiently at ontogenetically early phases including at the time of metamorphosis. 4. The CNS P0-like protein and the PNS P0 protein showed a difference in reactivity with lectins and anti-L2/HNK-1 antibodies, suggesting that the two proteins differ in some aspects of their carbohydrate structures.
Ambystoma↗
Relative properties and localizations of synaptic vesicle protein isoforms: the case of the synaptophysins.
Synaptophysins are abundant synaptic vesicle proteins present in two forms: synaptophysin, also referred to as synaptophysin I (abbreviated syp I), and synaptoporin, also referred to as synaptophysin II (abbreviated syp II). In the present study, the properties and localizations of syp I and syp II were investigated to shed light on their relative functions. Our results reveal that syp II, similar to syp I, is an abundant, N-glycosylated membrane protein that is part of a heteromultimeric complex in synaptic vesicle membranes. Cross-linking studies indicate that syp II is linked to a low-molecular-weight protein in this complex as has been observed before for syp I. Furthermore, after transfection into CHO cells, syp II, similar to syp I, is targeted to the receptor-mediated endocytosis pathway. Immunocytochemistry of rat brain sections reveals that syp II expression is highly heterogeneous, with high concentrations of syp II only in selected neuronal populations, whereas syp I is more homogeneously expressed in most nerve terminals. In general, nerve terminals expressing syp II also express syp I. In addition to high levels of syp II observed in selected neurons, a rostrocaudal gradient of syp II expression was observed in the cerebellar cortex. Immunoelectron microscopy confirmed that syp II is localized to synaptic vesicles. Immunoprecipitations of synaptic vesicles from rat brain with antibodies to syp I demonstrated that syp II is colocalized with syp I on the same vesicles. However, after detergent solubilization, no coimmunoprecipitations of the two proteins were observed, suggesting that they are not complexed with each other although they are on the same vesicles. Together our results demonstrate that syp I and syp II have similar properties and are present on the same synaptic vesicles but do not coassemble. The presence of the two proteins in the same nerve terminal suggests that they have similar but nonidentical functions and that the relative abundance of the two proteins may contribute to the functional heterogeneity of nerve terminals.
Animals↗