Rapid Phosphorylation of Phospholipase Cγ 1 by Brain-Derived Neurotrophic Factor and Neurotrophin-3 in Cultures of Embryonic Rat Cortical Neurons

Abstract: Phospholipase Cγ1 (PLC-γ1) is involved at an early step in signal transduction of many hormones and growth factors and catalyzes the hydrolysis of phosphatidylinositol (PI) 4,5-bisphosphate to diacylglycerol and inositol trisphosphate, two potent intracellular second messenger molecules. The transformation of PC12 cells into neuron-like cells induced by nerve growth factor is preceded by a rapid stimulation of PLC-γ1 phosphorylation and PI hydrolysis. The present study analyzed the effects of brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) on phosphorylation of PLC-γ1 in primary cultures of embryonic rat brain cells. BDNF and NT-3 stimulated the phosphorylation of PLC-γ1, followed by hydrolysis of PI. The stimulation of PLC-γ1 phosphorylation occurred within 20 s after addition of BDNF or NT-3 and lasted up to 30 min, with a peak after 4 min. ED₅₀ values were similar for BDNF and NT-3, with τ25 ng/ml. Phosphorylation of PLC-γ1 by BDNF and NT-3 was found in cultures from all major brain areas. K-252b, a compound known to inhibit selectively neurotrophin actions by interfering with the phosphorylation of trk -type neurotrophin receptors, prevented the BDNF- and NT-3-stimulated phosphorylation of PLC-γ1. Receptors of the trk type were coprecipitated with anti-PLC-γ1 antibodies. The presence of trkB mRNA in the cultures was substantiated by northern blot analysis. The action of BDNF and NT-3 seems to be neuron specific because no phosphorylation of PLC-γ1 was observed in cultures of nonneuronal brain cells. The results provide evidence that developing neurons of the cerebral cortex and other brain areas are responsive to BDNF and NT-3, and they indicate that the transduction mechanism of BDNF and NT-3 in the brain involves rapid phosphorylation of PLC-γ1 followed by PI hydrolysis.     

The GEF1 gene of Saccharomyces cerevisiae encodes an integral membrane protein; mutations in which have effects on respiration and iron-limited growth

We have isolated a new class of respiration-defective, i.e petite, mutants of the yeast Saccharomyces cerevisiae. Mutations in the GEF1 gene cause cells to grow slowly on rich media containing carbon sources utilized by respiration. This phenotype is suppressed by adding high concentrations of iron to the growth medium. Gef1 ^– mutants also fail to grow on a fermentable carbon source, glucose, when iron is reduced to low concentrations in the medium, suggesting that the GEF1 gene is required for efficient metabolism of iron during growth on fermentable as well as respired carbon sources. However, activity of the iron uptake system appears to be unaffected in gef1 ^– mutants. Fe(II) transporter activity and regulation is normal in gef1 ^– mutants. Fe(III) reductase induction during iron-limited growth is disrupted, but this appears to be a secondary effect of growth rate alterations. The wild-type GEF1 gene was cloned and sequenced; it encodes a protein of 779 amino acids, 13 possible transmembrane domains, and significant similarity to chloride channel proteins from fish and mammals, suggesting that GEF1 encodes an integral membrane protein. A gef1 ^– deletion mutation generated in vitro and introduced into wild-type haploid strains by gene transplacement was not lethal. Oxygen consumption by intact gef1 ^– cells and by mitochondrial fractions isolated from gef1 ^– mutants was reduced 25–50% relative to wild type, indicating that mitochondrial function is defective in these mutants. We suggest that GEF1 encodes a transport protein that is involved in intracellular iron metabolism.     

Association of the amino-terminal half of c-Src with focal adhesions alters their properties and is regulated by phosphorylation of tyrosine 527.

We have characterized the mechanism by which the subcellular distribution of c-Src is controlled by the phosphorylation of tyrosine 527. Mutation of this tyrosine dramatically redistributes c-Src from endosomal membranes to focal adhesions. Redistribution to focal adhesions occurs independently of kinase activity and cellular transformation. In cells lacking the regulatory kinase (CSK) that phosphorylates tyrosine 527, c-Src is also found predominantly in focal adhesions, confirming that phosphorylation of tyrosine 527 affects the location of c-Src inside the cell. The first 251 amino acids of c-Src are sufficient to allow association with focal adhesions, indicating that at least one signal for positioning c-Src in focal adhesions resides in the amino-terminal half. Point mutations and deletions in the first 251 amino acids of c-Src reveal that association with focal adhesions requires the myristylation site needed for membrane attachment, as well as the SH3 domain. Expression of the amino-terminal region alters both the structural and biochemical properties of focal adhesions. Focal adhesions containing this non-catalytic portion of c-Src are larger and exhibit increased levels of phosphotyrosine staining. Our results suggest that c-Src may regulate focal adhesions and cellular adhesion by a kinase-independent mechanism.     

Assignment of a locus for mouse lung tumor susceptibility to proximal Chromosome 19

Previous studies have hypothesized that at least three genetic loci contribute to differences in pulmonary adenoma susceptibility between mouse strains A/J and C57BL/6J. One gene that may confer susceptibility to lung tumorigenesis is the Kras protooncogene. To identify other relevant loci involved in this polygenic trait, we determined tumor multiplicity in 56 randomly chosen N-ethyl-N-nitrosourea-treated (A/J×C57BL/6J) N1×C57BL/6 backcross (AB6N2) progeny and correlated it with genotypes at 77 microsatellite markers spanning the genome. A correlation of lung tumor multiplicity phenotypes with genotypes of microsatellite markers on distal Chromosome (Chr) 6 in the Kras region (Pas1) was confirmed, and a new region on Chr 19 (designated Pas3) was identified that also contributes to susceptibility. Linkage analysis on Chr 19 with 270 AB6N2 mice localized the region flanked by D19Mit42 and D19Mit19 that is most closely associated with lung tumor susceptibility. The Pas3 locus may be an enhancer of the susceptibility locus on Chr 6.     

Immunochemical relationship of the major outer membrane protein of Rhodopseudomonas sphaeroides 2.4.1 to proteins of other photosynthetic bacteria.

Immunoblots of sodium dodecyl sulfate-polyacrylamide gels derived from outer membrane preparations of various strains of Rhodopseudomonas sphaeroides revealed polypeptides which cross-reacted with antibody directed against the major outer membrane protein of R. sphaeroides 2.4.1. Immunochemical quantitation of the major outer membrane protein of strain 2.4.1 showed approximately 5.5 x 10(4) molecules per cell whether cells were grown chemoheterotrophically or photoheterotrophically. Rhodospirillum rubrum outer membranes contained a cross-reactive protein, whereas the outer membranes derived from Rhodopseudomonas capsulata and Paracoccus denitrificans showed no cross-reaction with the antibody prepared against the major outer membrane protein from R. sphaeroides 2.4.1.     

The eel retina. Receptor classes and spectral mechanisms

Light and electron microscopy revealed that there are both rods and cones in the retina of the eel Anguilla rostrata. The rods predominate with a rod to cone ratio of 150:1. The spectral sensitivity of the dark-adapted eyecup ERG had a peak at about 520 nm and was well fit by a vitamin A2 nomogram pigment with a lambdamax = 520 nm. This agrees with the eel photopigment measurements of other investigators. This result implies that a single spectral mechanism--the rods--provides the input for the dark-adapted ERG. The spectral sensitivity of the ERG to flicker in the light-adapted eyecup preparation was shifted to longer wavelengths; it peaked at around 550 nm. However, there was evidence that this technique might not have completely eliminated rod intrusion. Rod responses were abolished in a bleached isolated retina preparation, in which it was shown that there were two classes of cone-like mechanisms, one with lambdamax of 550 nm and the other with lambdamax of less than 450 nm. Ganglion cell recording provided preliminary evidence for opponent-color processing. Horizontal cells were only of the L type with both rod and cone inputs.     

Specificity of cell surface virus receptors on radiation leukemia virus and radiation-induced thymic lymphomas.

We have developed a system for analysis of murine leukemic virus (MuLV) receptors on the surface of thymic lymphoma cells utilizing the fluorescence-activated cell sorter. The binding of fluoresceinated or rhodaminated MuLV to target cells showed saturation kinetics and was blocked by homologous MuLV, and bound MuLV had a polypeptide profile identical to that of input MuLV. Thymic lymphomas bound specifically the MuLV which induced them, whereas only 0.5 to 2% of normal thymocytes showed equivalent MuLV binding. Simultaneous binding of excess fluoresceinated RadLV and rhodaminated MCF-247 AKR virus to radiation leukemia virus-induced or spontaneous AKR thymic lymphomas demonstrated that even in the presence of both viruses the cells bound preferentially the inducing MuLV. Examination of the C57BL/Ka endogenous viruses showed that radiation leukemia virus-induced thymic lymphomas bind only thymotropic-leukemogenic radiation leukemia virus and not eco- or xenofibrotropic MuLV's. Thus, virus binding in this system involves only leukemogenic isolates of these retroviruses and implies a central role of this receptor-ligand interaction in the processes of leukemic transformation.     

Maternal discrimination of infant vocalizations in squirrel monkeys

Responses of mother squirrel monkeys to vocalizations of their own and other infants were examined to determine whether mothers could discriminate their infants on the basis of auditory cues. Thirty mothers, whose infants ranged in age from one to seven months were tested in three conditions in which their own infant, a different infant, and no infant served as the stimulus. Mothers were tested in an enclosed alleyway with opaque end panels behind which stimuli were placed. The quantity and quality of maternal responses clearly differed in the three conditions and indicated that mothers recognized their own infants. Differences in maternal vocalizations were the most pronounced. All but one type of vocalization increased in the own-infant condition; the exception, a high-pitched shrill, decreased. Mothers also spent more time near the stimulus and were more active when tested with their own infants.     

H-2 antigen expression: Loss in vitro,restoration in vivo,and correlation with cell-mediated cytotoxicity in a mouse lymphoma cell line

The susceptibility to cell-mediated cytolysis of cells of the recently developed C57BL/Ka(H-2 ^b ) lymphoma cell line, BL/VL₃, was investigated in allogeneic assays with thymus-dependent lymphocytes (T cells). Compared to EL4, the widely used C57BL/6(H-2 ^b ) lymphoma cell line, BL/VL₃ cells were found to be insensitive to T-cell-mediated lysis as detected by the use of⁵¹Crrelease methods. When used as immunogens in alloreactive combinations with BALB/c(H-2 ^d ) splenocytes as responder cells, BL/VL₃ cells failed to elicit sensitization. Serological tests showed that this cell line had profoundly reduced levels of H-2^b antigens on its surface. When BL/VL₃ cells were reinjected into C57BL/Ka and BALB/c mice, full recovery of H-2^b antigen expression at the cell surface was observed in both syngeneic and allogeneic hosts after only 11 days of in vivo growth. Concomitantly, they acquired the ability to induce cytotoxic responses in allogeneic T cells and became susceptible to their lytic activity. The expression of H-2 antigens on the surface of BL/VL₃ cells is a reversibly modulated function that depends on in vivo growth conditions and is lost in vitro in the absence of immunoselective pressure.