Studies on the turnover of mouse brain synaptosomal proteins

(l) The half-lives of the proteins of various fractions of whole mouse brain increase with increasing insolubility; the supernatant and hypotonic-extractable proteins had half-lives of about 13 days, whereas the membrane proteins solubilized with Triton X-100 and SLS had half-lives of about 18 days. The proteins of the subfractions of synaptosomes had half-lives ranging from 15 to 19 days; those in the cytoplasm had a half-life of 18·3 days, in the membranes, about 17 days and in the synaptic vesicles, 15·6 days. (2) Although the half-life of the synaptic vesicles was not significantly different from that of other synaptosomal subfractions, the vesicles exhibited a different protein pattern on acrylamide gels, an observation which implies that the proteins of the vesicles are qualitatively different from those of other synaptic membranes. (3) The uptake of labelled lysine into the cytoplasm of the synaptosomes of youg mice in vivo was very rapid. (4) The data derived from the relative specific radioactivities of synaptosomal fractions compared with their whole brain analogs support the contention that a sizeable fraction of the synaptosomal cytoplasmic protein was transported to the synapse by axoplasmic flow. The relative specific radioactivities of synaptosomal membrane and synaptic vesicle proteins rose much more quickly than the comparable activities for the cytoplasmic material, and the alternate possibility of synthesis in situ is discussed.     

Amino acid sequences of several Bacillus subtilis proteins modified by apparent guanylylation

Bacillus subtilis cell extracts, prepared at different times during growth, contained several proteins that were apparently guanylylated in vitro with [alpha-32P]-GTP. Four of the proteins were partially purified and the N-terminal amino acid sequences (13 to 20 residues) were determined. One sequence had 84% identity to Bacillus stearothermophilus triosephosphate isomerase, two were 100% identical to the predicted sequences of the B. subtilis ptsI and ptsH genes while no identity was found for the fourth sequence. This apparent guanylylation occurred with proteins involved in glucose metabolism, although the significance is unknown.     

Physical mapping and a new variant of Puroindoline b-2 genes in wheat

Puroindoline a and b proteins soften the endosperm of wheat kernels. When the underlying puroindoline genes are altered by mutation or are deleted, kernels become harder. Thus, puroindoline a and b (Pina and Pinb) play an important role in wheat quality and utilization. Recently, additional Pinb genes have been reported. In the present report, we provide corroborating coding and additional 5′ and 3′ flanking sequence for three Pinb variants: Pinb-2v1, Pinb-2v2, and Pinb-2v3. Additionally, a new Pinb variant, Pinb-2v4, is reported. All four variants were physically mapped using Chinese Spring (CS) diteolosomics, nullisomic–tetrasomics, and CS-Cheyenne disomic substitution lines. Results place Pinb-2v1 on 7DL, Pinb-2v2 on 7BL, Pinb-2v3 on 7B, and Pinb-2v4 on 7AL. Pinb-2v1 and Pinb-2v4 were present in all cvs. examined: CS, Cheyenne, Recital, Wichita and Winsome. Pinb-2v2 was present in CS and Recital; Pinb-2v3 was present in Cheyenne, Wichita, and Winsome. These results are not wholly consistent with prior research and additional studies will be required to reconcile discrepancies. The discovery of Pinb-2v4 and the mapping of all four variants will contribute to a better understanding of gene duplication events in wheat and their bearing on wheat kernel texture and grain utilization.     

Ca2+ and pH responses to sequential additions of mitogens in single 3T3 fibroblasts: correlations with DNA synthesis

The progression of Swiss 3T3 fibroblasts from the quiescent state (G0) through G1 to DNA synthesis in S phase generally requires the synergistic action of two mitogens. The main aim of this study was to compare systematically the early Ca2+ and pH responses in quiescent cells to all of the pair combinations of eight mitogens (bombesin, platelet-derived growth factor, vasopressin, prostaglandin F2 alpha, epidermal growth factor, 12-O-tetradecanoyl phorbol-13-acetate, insulin, 8-bromo-cAMP) with their subsequent effects on DNA synthesis. Each of the mitogens which caused inositol phosphate accumulation (bombesin, platelet-derived growth factor, vasopressin, prostaglandin F2 alpha) also activated Ca2+- and phospholipid-dependent protein kinase (protein kinase C) and generated both the Ca2+ and pH responses, although epidermal growth factor also generated the ionic responses without causing release of inositol phosphates or activation of protein kinase C. For sequential mitogen additions the ionic signals were measured in single cells as well as in cell populations to avoid ambiguities due to heterogeneity in the responses of the cells to the various mitogens. The modulating effects of the mitogens on the [Ca2+]i responses to subsequent mitogen additions varied widely, but detailed comparisons showed that the pattern of blocking effects could not be attributed solely to the effect of the first mitogen causing either maximal breakdown of phosphatidylinositol 4,5-bisphosphate or complete depletion of the intracellular Ca2+ pool or activation of protein kinase C. From these analyses it was concluded that the requirement for two mitogens for effective DNA synthesis could not be attributed to the summation to a critical threshold of either the ionic signals or phosphatidylinositol 4,5-bisphosphate breakdown, and that these responses are insufficient by themselves to cause the cells to progress to DNA synthesis in S phase.