Neto2 Interacts with the Scaffolding Protein GRIP and Regulates Synaptic Abundance of Kainate Receptors

Kainate receptors (KARs) are a class of ionotropic glutamate receptors that are expressed throughout the central nervous system. The function and subcellular localization of KARs are tightly regulated by accessory proteins. We have previously identified the single-pass transmembrane proteins, Neto1 and Neto2, to be associated with native KARs. In the hippocampus, Neto1, but not Neto2, controls the abundance and modulates the kinetics of postsynaptic KARs. Here we evaluated whether Neto2 regulates synaptic KAR levels in the cerebellum where Neto1 expression is limited to the deep cerebellar nuclei. In the cerebellum, where Neto2 is present abundantly, we found a ∼40% decrease in GluK2-KARs at the postsynaptic density (PSD) of Neto2-null mice. No change, however, was observed in total level of GluK2-KARs, thereby suggesting a critical role of Neto2 on the synaptic localization of cerebellar KARs. The presence of a putative class II PDZ binding motif on Neto2 led us to also investigate whether it interacts with PDZ domain-containing proteins previously implicated in regulating synaptic abundance of KARs. We identified a PDZ-dependent interaction between Neto2 and the scaffolding protein GRIP. Furthermore, coexpression of Neto2 significantly increased the amount of GRIP associated with GluK2, suggesting that Neto2 may promote and/or stabilize GluK2:GRIP interactions. Our results demonstrate that Neto2, like Neto1, is an important auxiliary protein for modulating the synaptic levels of KARs. Moreover, we propose that the interactions of Neto1/2 with various scaffolding proteins is a critical mechanism by which KARs are stabilized at diverse synapses.     

Abnormalities of the ear associated with exencephaly in mouse fetuses induced by maternal exposure to cadmium

Exencephaly was induced in mouse fetuses by maternal injection of cadmium chloride (CdCl2) on day 7 of gestation. The heads of exencephalic, nonexencephalic experimental, and control fetuses were embedded in paraffin and sections were stained with hematoxylin and eosin. Compared to those of controls, the ears of the exencephalic fetuses were smaller (microtia) and low set. The meatal plug representing the external auditory canal was thick, variously branched, and often directed inferiorly. Usually, there were just two ossicles. The stapedial artery, facial nerve, and stapedius muscle were hypoplastic; the tensor tympani was small or absent. There were 1.0 to 2.0 turns of the cochlea in contrast to 2.5 turns in the controls. The organ of Corti was underdifferentiated; the spiral ganglion had fewer cells. In the control, the long axes of the anterior and posterior semicircular ducts were at right angles to each other and in vertical planes, but in the exencephalics, they tended more laterally towards the horizontal plane. The differentiation of the cristae ampullares and maculae was also severely affected. In several specimens, the entire membranous labyrinth had been distended; these labyrinths also had unusual epithelial infoldings. In cadmium-treated nonexencephalic fetuses, the external ears were normal and appropriate to the body size; five of them were examined histologically; in all, the five middle ear contents were hypoplastic; in three, the cochlea had a maximum of two turns and the organ of Corti, crista ampullaris, and macula were hypoplastic. By an analogy to abnormalities of mutants with neural tube defects, it is suggested that the exencephaly induced by cadmium might affect the differentiation of the ear. Partial involvement of the ear in nonexencephalic experimental embryos may be the result of direct action of cadmium during critical stages of development.     

Embryonal carcinoma-derived growth factor activates protein kinase C in vivo and in vitro.

We have recently reported that a polypeptide mitogen, the embryonal carcinoma-derived growth factor (ECDGF), induces phosphorylation of the epidermal growth factor (EGF) receptor in intact C3H 10T 1/2 mouse fibroblasts with concomittant loss of high affinity EGF binding sites. This phenomenon appears to be mediated through an activation of protein kinase C. Several groups have described an acidic 80,000 dalton protein substrate of protein kinase C. In this paper, we demonstrate that the addition of ECDGF or the phorbol ester TPA to intact C3H 10T 1/2 cells results in the enhanced phosphorylation of this 80 kd protein in vivo. Furthermore, this response is demonstrable in vitro. Thus the addition of ECDGF, the phorbol ester TPA, protein kinase C or phosphoinositidase C to crude membranes prepared from C3H 10T 1/2 cells resulted in the enhanced phosphorylation of this protein. Data obtained by phosphopeptide mapping of the 80 kd protein show that the ECDGF-induced activation of protein kinase C in our membrane preparations is comparable with that obtained in vivo. The availability of an in vitro system in which this response is preserved should now allow a detailed biochemical analysis of the steps between binding of a mitogen to its receptor and the activation of protein kinase C.     

Pathogenesis of exencephaly and cranioschisis induced in the rat after neural tube closure: role of the mesenchyme

Exencephaly was induced after neural tube closure by administration of a single dose of 15 mg/kg cyclophosphamide (CPA) to pregnant Wistar rats. Embryos were collected at 6, 8, and 10 hr and then at 24-hr intervals until day 17 of gestation and processed for electronmicroscopy. Sections cut at the level of foramen of Monro and at the otic vesicle level were examined and compared with similarly processed and age-matched control embryonic tissues. The earliest changes in the CPA-treated embryonic mesenchyme (ME) included a relative reduction in polyribosomes and an accumulation of cellular debris in apparently normal cells and in the extracellular space (ECS). While dead cells were disintegrating in the ECS, numerous cells were engaged in phagocytosis and digestion of fragments of dead cells. Continued cell loss and inhibition of cell proliferation lead to a marked increase in ECS and loss of intercellular contacts. At 10 hr postinjection, these changes were accentuated. By day 13, the capillaries in the ME were found to have their endothelium attenuated; they also had no pericyte association. Unlike in controls, the CPA embryos never developed a proper primordium of the skull vault. Differentiation of the poorly organised ME cells was slow, and glycogen appeared in them late and persisted until day 17. The endoplasmic reticulum was dilated; lipid and lysosomes accumulated, and matrix secretion was inhibited. Macrophages were numerous. The capillaries proliferated and possessed intraluminal cytoplasmic flaps resembling capillaries in the controls at earlier stages of development. The ECS in the ME became edematous while blebs developed subcutaneously. The weak neuroepithelium had several cavities that communicated internally with the ventricle and externally with the blebs. Cell death, inhibition of cell proliferation, and failure of the appearance of skull vault primordium resulted in poor support to the NE. The mounting intraventricular pressure possibly lead to a breakdown of the unsupported NE, resulting in reopening of the neural tube.     

Changes in the Levels of Free Amino Acids in Various Regions of Cuscuta during Growth

The angiospermous plant parasite Cuscuta derives reduced carbonand nitrogen compounds primarily from its host. Free amino acidsalong Cuscuta vines in three zones, viz., 0 to 5 cm, 5 to 15cm, and 15 to 30 cm, which in a broad sense represent the regionof cell division, cell elongation and differentiation and vasculartissue differentiation respectively, were quantitatively estimated.The free amino acid content was the highest in the 0 to 5 cmregion and progressively decreased along the posterior regionsof the vine. The haustorial region showed the lowest contentof free amino acids. In general, the free amino acid contentin samples collected at 7 p.m. was found to be higher than thatin the samples collected at 7 a.m. Three basic amino acids,histidine, the uncommon amino acid -hydroxyarginine, and arginineconstituted more than 50% of the total free amino acids in allthe zones studied except the haustorial region. Aspartic acidand glutamic acid constituted the major portion in the acidicand neutral fraction of amino acids. Glutamine, asparagine,threonine, and serine were eluted together and occurred in substantialamounts. -Hydroxyarginine constituted the largest fraction inthe cut end exudate of Cuscuta and presumably appeared to bethe major form of transport amino acid. -Hydroxyarginine wasalso a major constituent of the basic amino acids in Cuscutavines parasitizing host plants from widely separated families,suggesting that this amino acid is a biosynthetic product ofthe parasite rather than that of the hosts. Also, U-¹⁴C argininewas converted to -hydroxyarginine by cut Cuscuta vines, suggestingthat -hydroxyarginine is synthesized de novo from arginine byCuscuta. (Received March 30, 1987; Accepted June 7, 1988)     

Intracellular signalling pathways involved in mesoderm induction by FGF.

We have examined the possible role of two signal transducing mechanisms, tyrosine phosphorylation and activation of protein kinase C (PKC), during fibroblast growth factor (FGF)-induced mesoderm induction in Xenopus. Tyrosine phosphorylation was examined through the use of a monoclonal anti-phosphotyrosine antibody. This antibody was shown to recognize the FGF receptor crosslinked to radioiodinated FGF. We also studied the response of Xenopus ectodermal explants to sodium orthovanadate, a compound that has been shown to elevate intracellular phosphotyrosine levels. Thirty percent of explants cultured in 100 microM vanadate were induced. In addition, vanadate synergized with FGF to give inductions that were more dorsal in nature than either vanadate or FGF alone. The role of PKC was evaluated by measuring PKC activity during mesoderm induction by FGF and by examining the effect of the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) on explants. TPA did not induce mesoderm, however, activation of PKC was detected in FGF-treated explants. Therefore, activation of the PKC pathway alone is not sufficient for mesoderm induction. Simultaneous treatment with TPA and FGF resulted in a significant inhibition of mesoderm induction by FGF, suggesting that activation of PKC could be part of a negative feedback mechanism. In contrast, TPA had no effect on induction by activin A.     

Dodecylglycerol. A new type of antibacterial agent which stimulates autolysin activity in Streptococcus faecium ATCC 9790

Dodecylglycerol has a minimum inhibitory concentration of 4 micrograms/ml compared to 9 micrograms/ml for monolaurin (dodecanoylglycerol) with Streptococcus faecium ATCC 9790 as the test organism. The greater potency of dodecylglycerol can be correlated to its greater retention by the cell. Gram-positive bacteria were more susceptible than Gram-negative bacteria to dodecylglycerol. The antibacterial action of dodecylglycerol is not through the physical dissolution of cell walls, but rather as an enzymatic effector. The autolysin activity of whole cells of S. faecium was greatly stimulated by dodecylglycerol. The stimulation of autolytic activity and inhibition of growth respond in parallel to different concentrations of dodecylglycerol, to dodecylglycerol versus some poorer effector such as monolaurin or a glycerol alkyl ether with a longer or shorter fatty alkyl side chain than dodecanol, and to the antagonistic effects of diphosphatidlyglycerol. This close relationship implies that the stimulation of autolysin activity could be a primary, but not necessarily the only, mechanism by which dodecylglycerol and related compounds exert their antibacterial activity. However, the autolysin activity is not stimulated by a direct interaction between the enzyme and dodecylglycerol. A non-wall entity, such as a proteinase, has been implicated as an intermediary (Ved, H. S., Gustow, E., and Pieringer, R. A. (1984) J. Biol. Chem. 259, 8122-8124).