Recent evidence indicates that L-glutamate is taken up into synaptic vesicles in an ATP-dependent manner, supporting the notion that synaptic vesicles may be involved in glutamate synaptic transmission. In this study, we further characterized the ATP-dependent vesicular uptake of glutamate. Evidence is provided that a Mg-ATPase, not Ca-ATPase, is responsible for the ATP hydrolysis coupled to the glutamate uptake. The ATP-dependent glutamate uptake was inhibited by agents known to dissipate the electrochemical proton gradient across the membrane of chromaffin granules. Hence, it is suggested that the vesicular uptake of glutamate is driven by electrochemical proton gradients generated by the Mg-ATPase. Of particular interest is the finding that the ATP-dependent glutamate uptake is markedly stimulated by chloride over a physiologically relevant, millimolar concentration range, suggesting an important role of intranerve terminal chloride in the accumulation of glutamate in synaptic vesicles. The vesicular glutamate translocator is highly specific for L-glutamate, and failed to interact with aspartate, its related agents, and most of the glutamate analogs tested. It is proposed that this vesicular translocator plays a crucial role in determining the fate of glutamate as a neurotransmitter.

A B S T R A C T When the fatigue life N f of a specimen of 10 mm in thickness is longer than 10 8 cycles, the average fatigue crack growth rate is much less than the lattice spacing (~0.1 Å or 0.01 nm) that is 10 −11 to 10 −12 m/cycle. In the early stage of the fatigue process, the crack growth rate should be much less than the average growth rate, and accordingly we cannot assume that crack growth occurs cycle by cycle.In this paper, possible mechanisms for extremely high cycle fatigue are discussed. Of some possible mechanisms, a special focus was put on a newly found particular fatigue fracture morphology in the vicinity of the fracture origin (non-metallic inclusions) of a heat-treated alloy steel, SCM435, which was tested to NÁ10 8 . The particular morphology observed by SEM and AFM was presumed to be influenced by the hydrogen around inclusions. The predictions of the fatigue limit by the ǰarea parameter model are~10% unconservative for a fatigue life of N f =~10 8 , though it successfully predicts the conventional fatigue limit defined for N=10 7 . Thus, the fatigue failure for NÁ10 8 is presumed to be caused by a mechanism which induces breaking or releasing of the fatigue crack closure phenomenon in small cracks.In the vicinity of a non-metallic inclusion at the fracture origin, a dark area was always observed inside the fish-eye mark for those specimens with a long fatigue life. Specimens with a short fatigue life of N f =~10 5 do not have such a dark area in the fish-eye mark. SEM and AFM observations revealed that the dark area has a rough surface quite different from the usual fatigue fracture surface in a martensite lath structure.Considering the high sensitivity of high-strength steels to a hydrogen environment and the high hydrogen content around inclusions, it may be concluded that the extremely high cycle fatigue failure of high-strength steels from non-metallic inclusions is caused by environmental effects, e.g. hydrogen embrittlement coupled with fatigue.

Neutron scattering experiments have been carried out on the heavy fermion antiferromagnetic (AFM) superconductor CePt 3 Si with T N = 2.2 K and T SC = 0.75 K. We observed clear AFM Bragg reflections with Q 0 = (001/2) below and above T SC , indicating microscopic coexistence of AFM order and heavy fermion superconductivity. The AFM structure, of two interleaved ferromagnetic sublattices of local Ce 4f moments, has inversion symmetry under simultaneous space-time reversal. However, hybridization with Pt and Si breaks this degeneracy and a combination of these two competing effects may be relevant to an understanding of the simultaneous occurrence of superconductivity and AFM order. The observed magnetic moment 0.16(1) µ B /Ce is strongly reduced from the Curie-Weiss effective moment 2.54 µ B /Ce. Clear crystal field excitations at 1 and 24 meV were observed. The magnetic susceptibility can be well explained in a level scheme assuming the 7 ground state, 6 and 7 first and second excited states, respectively.

We have studied the superconducting property by measuring the electrical resistivity under the magnetic field and pressure for CePt 3 Si, which is the first antiferromagnetic heavy fermion superconductor without inversion symmetry in its tetragonal crystal structure. The upper critical field H c2 ð0Þ is found to be approximately isotropic: H c2 ð0Þ ¼ 2:7 T for H k ½100 and 3.2 T for H k ½001. The Néel temperature T N ¼ 2:3 K and the zero resistivity superconducting transition temperature T sc ' 0:65 K decrease markedly with increasing pressure, suggesting that the present superconductivity is correlated to the antiferromagnetic state.A recently discovered superconductor CePt 3 Si is unique, possessing a few characteristics. 1) Superconductivity with the transition temperature T sc ¼ 0:75 K is realized in the long-range antiferromagnetic state with the Néel temperature T N ¼ 2:2 K. Both transitions were observed clearly in the specific heat measurement. The electronic specific heat coefficient is large, 300-400 mJ/K 2 Ámol. In the previous cerium-based heavy fermion superconductors including a prototype superconductor CeCu 2 Si 2 and a quasi-two-dimensional superconductor CeCoIn 5 with a large value of 1000 mJ/K 2 Ámol, superconductivity occurs in the nonmagnetic state or the antiferromagnetically spin-fluctuating state. 2-4) Another example is CeIn 3 under pressure. 4-6) At ambient pressure, the Néel temperature in CeIn 3 is T N ¼ 10 K and the ordered moment is 0.5 B /Ce. With increasing pressure P, T N decreases smoothly as a function of pressure and becomes zero at around a critical pressure P c ' 2:7 GPa where superconductivity sets in below 0.2 K.Moreover, it is noted that CePt 3 Si is the first heavy fermion superconductor lacking a center of symmetry. The crystal structure of CePt 3 Si is tetragonal, space group P4mm (No. 99) with a ¼ 4:072 # A and c ¼ 5:442 # A. The existence of inversion symmetry in the crystal structure is believed to be a favorable factor for superconductivity. The absence of superconductivity in, for example, ferromagnet MnSi was tentatively attributed to the lack of inversion symmetry in its crystal structure. 7) Theoretically it was, however, argued that spin-triplet pairing is not entirely excluded in MnSi and CePt 3 Si because the lack of inversion symmetry reduces the effect of paramagnetic limiting for spin-singlet pairing. 8) In fact we have very recently observed superconductivity in the vicinity of a critical pressure for ferromagnet UIr without inversion symmetry. 9) The relationship between superconductivity and lack of inversion symmetry is the most crucial issue to be clarified at present.We have studied an effect of magnetic field and pressure on the superconducting state for a single crystal with high quality. The electrical resistivity has been measured to investigate the anisotropy of the upper critical field and the relationship between antiferromagnetic ordering and superconductivity.Single crystals of CePt 3 Si and non-4f reference compound LaPt 3 Si were grown by t...

The subcellular distribution of Proteins la and lb, two proteins which serve as specific substrates for protein kinases present in mammalian brain, was studied in the dog cerebral cortex. Proteins Ia and Ib were found to be most highly enriched in synaptic vesicle fractions ; they were also present in postsynaptic density and synaptic membrane fractions in significant amounts, Proteins la and Ib present in the synaptic vesicle fraction appear to be similar, if not identical, to those present in the postsynaptic density fraction as judged by several criteria : (a) the ability to serve as substrate for cAMP-dependent protein kinase, (b) electrophoretic mobility in the presence of sodium dodecyl sulfate, (c) extractability with NH4 C1 or EGTA, and (d) fragmentation to electrophoretically similar peptides by a purified Staphylococcus aureus protease . In addition, the postsynaptic density fraction has been found to contain cAMP-dependent Protein Ia and Protein Ib kinase activity . The subcellular localization of Proteins la and Ib suggests a role for these proteins in the physiology of the synapse .

Glucose is the major source of brain energy and is essential for maintaining normal brain and neuronal function. Hypoglycemia causes impaired synaptic transmission. This occurs even before significant reduction in global cellular ATP concentration, and relationships among glycolysis, ATP supply, and synaptic transmission are not well understood. We demonstrate that the glycolytic enzymes glyceraldehyde phosphate dehydrogenase (GAPDH) and 3-phosphoglycerate kinase (3-PGK) are enriched in synaptic vesicles, forming a functional complex, and that synaptic vesicles are capable of accumulating the excitatory neurotransmitter glutamate by harnessing ATP produced by vesiclebound GAPDH/3-PGK at the expense of their substrates. The GAPDH inhibitor iodoacetate suppressed GAPDH/ 3-PGK-dependent, but not exogenous ATP-dependent, Glycolysis plays a vital role in maintaining normal brain function. Glucose is known to serve as the major substrate for cerebral energy under normal conditions (1). Recent evidence suggests a direct correlation between glucose utilization and cognitive function (2). Reduction of glucose levels results in pathophysiological states and abnormal electrophysiological activity; however, this occurs long before significant alteration in tissue ATP levels is detected (3-7). Substitution of pyruvate for glucose does not support normal evoked neuronal activity, although tissue ATP level returns to normal (8 -10). Abnormal synaptic transmission caused by hypoglycemia occurs in part if not entirely by a presynaptic mechanism (7,11,12). Fleck et al. (7) have shown that substantial reduction of extracellular glucose results in a decrease in stimulus-evoked Glu release, with no changes in ATP levels. These studies together suggest that glycolysis or glycolytic intermediate(s) are necessary for normal synaptic transmission independent of global cellular ATP levels.In an attempt to reveal the underlying mechanism of hypoglycemia-induced aberrant synaptic transmission, we previously explored the possibility that glycolytic intermediates could modify proteins localized in the nerve ending (13,14). 3-Phosphoglycerate (3-PG) 1 was demonstrated to stimulate phosphorylation of 155-and 72-kDa proteins. The latter was identified as glucose-1,6-bisphosphate synthetase, and 1,3-bisphosphoglycerate (1,3-BPG) was found to serve as the direct substrate for phosphorylation of this enzyme, by donating 1-phosphate. Both of these phosphorylated proteins are enriched in the synaptosomal (nerve ending preparation) as well as cell body soluble fractions, but the significance of these modifications in synaptic transmission remains unclear.In this paper, we show that the glycolytic intermediate 1,3-BPG forms an acyl-enzyme intermediate with vesicle-bound glyceraldehyde phosphate dehydrogenase (GAPDH), that vesicle-bound GAPDH exists in a complex with 3-phosphoglycerate kinase (3-PGK), and that activation of vesicle-associated GAPDH and 3-PGK is sufficient to support vesicular uptake of Glu. Glutamate is now recognized as the major ex...

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Glutamate plays an important metabolic role in virtually every vertebrate cell. In particular, glutamate is the most common excitatory neurotransmitter in the vertebrate central nervous system. As such, the mechanism by which glutamate is diverted from its normal metabolic activities toward its role as a neurotransmitter has, in recent years, been systematically investigated. In glutamatergic nerve endings, synaptic vesicles accumulate and store a proportion of the cellular glutamate pool and, in response to appropriate signals, release glutamate into the synaptic cleft by exocytosis. Glutamate accumulation is accomplished by virtue of a glutamate uptake system present in the synaptic vesicle membrane. The uptake system consists of a transport protein, remarkably specific for glutamate, and a vacuolar-type H+-ATPase, which provides the coupling between ATP hydrolysis and glutamate transport. The precise manner in which the glutamate transporter and H+-ATPase operate is currently the subject of debate. Recent data relevant to this debate are reviewed in this article. Additionally, pharmacological agents thought to specifically interact with the vesicular glutamate transporter are discussed. Finally, a newly discovered, endogenous inhibitor of vesicular uptake, inhibitory protein factor (IPF), is discussed with some speculations as to its potential role as a presynaptic modulator of neurotransmission.