HE TURNOVER RATE OF TUBULIN IN RAT BRAIN

The turnover rate of tubulin in rat brain was determined from the decay in specific radioactivity of the protein after pulse-labeling. When precursors were administered by a parenteral route, the shortest half-life, 9.8 days, was obtained with [¹⁴C]NaHCO₃; the longer half-lives obtained with [U-¹⁴C]glucose or [4,5-³H]leucine suggest significant reutilization of label. Furthermore, with leucine as precursor maximal specific radioactivity of tubulin was not obtained until eight days after administration of label. Labeling and decay kinetics obtained with [4,5-³H]leucine were markedly different when the isotope was administered directly into the lateral ventricle. The difference between the turnover rates of the -α and β subunits of tubulin purified by means of high resolution polyacrylamide gel electrophoresis was not statistically significant. A half-life for tubulin of 6.2 days was measured by continuous intravenous infusion of [U-¹⁴C]tyrosine.     

Adenine nucleotide pool size,adenine nucleotide translocase activity,and respiratory activity in newborn rabbit liver mitochondria

The adenine nucleotide content (ATP+ADP+AMP) of newborn rabbit liver mitochondria was 6.0±0.5 nmol/mg mitochondrial protein at birth, increased rapidly to 14.5±1.7 nmol/mg protein by 2 h postnatal, peaked at 6 h, then decreased gradually to 7.8±0.6 nmol/mg protein by 4 days postnatal. There was a strong positive correlation (r=0.82) between the total adenine nucleotide pool size and adenine nucleotide translocase activity in these mitochondria. In contrast, glutamate + malate-supported State 3 respiratory rates remained constant from birth through the first week of life. State 4 rates also remained constant, as did the respiratory control index and uncoupled respiratory rates. The following conclusions are suggested: (1) The maximum rate of translocase activity is limited by the intramitochondrial adenine nucleotide pool size. (2) In newborn rabbit liver mitochondria, the State 3 respiratory rate is not limited by either the adenine pool size or the maximum capacity for translocase-mediated adenine exchange. (3) In contrast to rat, rabbit liver mitochondria are fully functional at birth with regard to respiratory rates and oxidative phosphorylation. (4) The rapid postnatal accumulation of adenine nucleotides by liver mitochondria, now documented in two species, may be a general characteristic of normal metabolic adjustment in neonatal mammals.     

Bimodal effect of phorbol ester on B cell activation. Implication for the role of protein kinase C.

The role of protein kinase C PKC in B cell activation is controversial. These studies were undertaken to determine whether protein kinase C has a stimulatory or inhibitory role in B cell activation. We found that treatment of B cells for a short period of time (30 min) with the PKC activator phorbol 12,13-dibutyrate (PDBU) primed the cells for enhanced proliferative responses to anti-immunoglobulin (anti-Ig) antibody whereas treatment for a longer period of time (3 h or more) resulted in suppression of proliferation. The enhanced proliferative response to treatment of B cells with PDBU for short periods of time was associated with inhibition of anti-Ig-stimulated increases in phosphatidyl 4,5-bisphosphate (PIP2) hydrolysis and inhibition of increases in [Ca2+]i, indicating that activation of PKC per se might be sufficient for enhancing B cell activation. The time-dependent effect of phorbol esters on the inhibition of B cell proliferation was found to be closely correlated with the kinetics of disappearance of PKC as measured by Western blot and by enzymatic activity but not with inhibition of [Ca2+]i and PIP2. These data demonstrate a bimodal time-dependent effect of PDBU on B cell activation and suggest that (a) the inhibitory effect of phorbol ester on anti-Ig-induced proliferation may be due to the disappearance of PKC rather than to the inhibition of PIP2 and Ca2+; and (b) the early activation of PKC is a stimulatory rather than an inhibitory signal in the induction of B lymphocyte proliferation by anti-Ig.     

Evaluation of human monoclonal antibody (2-139-1) in cutaneous melanocytic neoplasms in fixed tissue sections.

Despite the growing list of xenogeneic monoclonal antibodies (MAb) that recognize malignant melanoma-associated antigens (MAA) in formalin-fixed, paraffin-embedded tissue, none has been able to detect epitopes found in malignant melanomas and not in melanocytic nevi. A human MAb, 2-139-1, that showed promise in this regard was evaluated against 85 melanocytic neoplasms, including malignant melanoma and histological simulators, particularly Spitz's nevus. MAb 2-139-1 stained 18 (53%) of 34 melanomas, eight (57%) of 14 dysplastic nevi, six (38%) of 16 Spitz's nevi, and three (14%) of 21 banal nevi, which included three small congenital nevi. We observed a significant increasing trend in reactivity (% positive cells x intensity) associated with the potential for malignancy (p for linear trend = 0.002). We conclude that human MAb 2-139-1 is applicable to the study of melanocytic neoplasms in routinely processed tissue. Although the ability of this MAb to separate benign from malignant cells is not absolute, our results suggest that the expression of the 2-139-1 epitope may be an early event in melanocytic tumor progression.     

Cross-linkage of Ly-6A/E induces Ca2+ translocation in the absence of phosphatidylinositol turnover and mediates proliferation of normal murine B lymphocytes

Ly-6A/E is a phosphatidylinositol (PI)-linked membrane protein whose expression is induced or upregulated on normal murine T and B cells by IFN-gamma. Cross-linkage of Ly-6A/E expressed on normal murine T cells stimulates Ca2+ translocation, and in the presence of a protein kinase C (PKC) activator, lymphokine secretion, and cellular proliferation. Utilizing an anti-Ly-6A/E mAb, we studied the effect of cross-linking Ly-6A/E on IFN-gamma-treated resting B cells, for Ca2+ translocation, PI turnover, and cellular proliferation. Since these events are known to be stimulated by cross-linkage of B cell membrane (m)Ig, we compared the changes mediated through these respective membrane proteins. We show that cross-linkage of B cell Ly-6A/E stimulates a large, rapid, and sustained increase in the concentration of intracellular free calcium ([Ca2+]i) comparable in magnitude, though somewhat delayed, relative to that observed after cross-linking of mIg. Cross-linkage of B cell Ly-6A/E does not, however, stimulate detectable PI turnover, in contrast to PI turnover induced by ligation of mIg. Both the Ly-6A/E- and mIg-mediated increase in [Ca2+]i occur through mobilization of internal Ca2+ stores as well as entry of Ca2+ into the cell from the extracellular compartment. Ly-6A/E-mediated Ca2+ translocation appears to be under the regulation of PKC in that short term pretreatment of B cells with the PKC activator, PMA, inhibits the Ly-6A/E- as well as the mIg-mediated increase in [Ca2+]i, whereas prolonged exposure to PMA, under conditions that lead to depletion of PKC, results in an augmentation in Ca2+ translocation after ligation of either Ly-6A/E or mIg. Co-capping studies indicate that Ly-6A/E and mIg cap independently in the B cell membrane, thus suggesting that the Ly-6A/E-induced effects on Ca2+ translocation are not mediated through simultaneous modulation of mIg. Anti-Ly6A/E, by itself, does not stimulate an increase in [3H]thymidine incorporation by IFN-gamma-treated resting B cells, but induces a striking increase in the presence of PMA. By contrast, anti-Ig by itself stimulates significant increases in [3H]thymidine incorporation that is inhibited by PMA. Thus, Ly-6A/E is a potent mediator of B cell activation that may use a signal transduction system in quiescent B cells that is distinct from that of the Ag receptor.     

Tyrosine kinase-regulated and inositol phosphate-independent Ca2+ elevation and mobilization in T cells.

Perturbation of the T cell antigen-specific receptor leads to a series of signaling events that includes a rapid increase in phosphoinositide hydrolysis, intracellular Ca2+, and tyrosine phosphorylation. We have examined the function of tyrosine phosphorylation in isolation by introducing the v-src tyrosine kinase into a T cell hybridoma. T cell receptor-mediated increases in phosphoinositide hydrolysis and, in particular the generation of inositol 1,4,5-trisphosphate, were comparable between v-src+ and v-src- cells. Unexpectedly, the v-src+ cells exhibited spontaneously elevated intracellular Ca2+ and exaggerated Ca2+ increases when stimulated via the T cell receptor. The enhanced Ca2+ response was not due to tyrosine phosphorylation of the T cell receptor itself, since the phenotype was evident in T cell receptor zeta chain-/v-src+ cells as well. These results demonstrate that an active protein tyrosine kinase can markedly affect intracellular Ca2+ handling by a process independent of inositol 1,4,5-trisphosphate production and T cell receptor tyrosine phosphorylation and raise the possibility that tyrosine kinases may directly regulate T cell receptor-mediated changes in intracellular Ca2+.     

Characterization of Enkephalin Release from Rat Striatum

Abstract: Using antisera specific for methionine- and leucine-enkephalin, we studied the characteristics of the release of these peptides from rat striatal slices. Only 2–3% of the total tissue stores of enkephalin could be released by potassium depolarization; similar percentages were released from globus pallidus, thalamus, and nucleus accumbens. Enkephalin release from hippocampus could not be detected. The striatal release of both enkephalins was affected similarly by changes in potassium and calcium levels in the superfusion medium. Lithium has no effect on either basal or potassium-stimulated release; tyr-arg did not affect basal release of either peptide. Striatal enkephalin levels were stable during the short-term incubation periods used in these experiments.     

The c1 repressor of bacteriophage P1: I. Isolation of the c1 protein and determination of the P1 DNA region to which it Binds

A bacteriophage P1-specific DNA binding protein has been partially purified from P1-infected Escherichia coli and identified as the P1c1 repressor. This protein is absent from non-suppressing cells infected with a P1c1 amber mutant. The binding activity of the protein isolated from cells infected with a c1ts mutant is thermolabile in vitro, so the repressor protein is the product of the c1 gene. Studies on P1 DNA fragments generated by restriction endonuclease digestion indicate that the c1 repressor binds preferentially in vitro at a site or sites located close to the c1 gene itself.     

Induction of nitrate reductase and membrane cytochromes in wild type and chlorate-resistant Paracoccus denitrificans

An experimental system has been devised for induction of nitrate reductase in suspensions of wild type Paracoccus denitrificans incubated with limited aeration in the presence of azide, nitrate or nitrite. Azide promoted maximum synthesis of enzyme, accompanied by formation of excess b-type cytochrome; the level of enzyme attained with nitrate was less and c-type cytochrome predominated in the membrane. The nitrate reductase was solubilized with deoxycholate from membranes of azide-induced cells and was identified as a major polypeptide M _r =150,000 by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Mutants strains lacking nitrate reductase activity were isolated on the basis of resistance to chlorate and mutant M-1 was examined in detail. When incubated in the cell suspension system M-1 formed a membrane protein M _r =150,000 similar to that attributed to nitrate reductase in the wild type. Maximum formation of the protein by M-1 occurred without inducer and it was accompanied by synthesis of excess b-type cytochrome. The observations with wild type and M-1 indicate that nitrate reductase protein and b-type cytochrome are coregulated and that the active enzyme has a role in regulating its own synthesis.Non-standard Abbreviations SDS sodium dodecyl sulphate - PAGE polyacrylamide gel electrophoresis - DOC sodlum deoxycholate