Acetoacetyl--coenzyme A thiolase in brain, liver, and kidney during maturation of the rat.

The development of thiolase in various rat tissues from 7 days before birth to adulthood was studied. Enzyme activities in brain, liver, and kidney during the perinatal period reflect the nutritional environment, whereas those in the adult tissues do not.

Long-term stimulation of cAMP production in LLC-PK1 pig kidney epithelial cells by salmon calcitonin or a photoactivatable analogue of vasopressin.

A photoreactive analogue of vasopressin, [1-(3-mercapto)propionic acid, 8-(N6-4-azidophenylamidino)lysine]-vasopressin, was compared to salmon calcitonin and [8-arginine]-vasopressin with respect to stimulation of cAMP synthesis in the LLC-PK1 pig kidney epithelial cell line. Without photoactivation, the vasopressin analogue-elicited responses were identical to those induced by vasopressin, in that cAMP synthesis returned to the basal, unstimulated level about 4 h after hormonal treatment. In contrast, the levels of activation of cAMP-dependent protein kinase induced by salmon calcitonin returned to basal approx. 12 h after hormone addition. When activated by ultraviolet irradiation, the vasopressin analogue induced 'permanent' stimulation of adenylate cyclase, whereby cAMP production could be detected even 12.5 h after treatment. Both salmon calcitonin and the photoactivated vasopressin analogue inhibited growth of LLC-PK1 cells, in contrast to vasopressin or the nonactivated analogue. Growth inhibition appeared to be a consequence of the prolonged stimulation of adenylate cyclase. This conclusion was supported by the fact that a LLC-PK1 cell mutant in cAMP-dependent protein kinase was resistant to growth inhibition by salmon calcitonin and activated vasopressin analogue. The results imply that the cAMP-dependent protein kinase is the mediator of the hormone-stimulated growth inhibition.

In vitro transcription of vitellogenin sequences on chick liver chromatin.

The in vitro transcription of chick liver chromatin before and after estrogen treatment was studied. Transcription was by endogenous as well as homologous exogenous RNA polymerase II and the products were analyzed for size and specific vitellogenin sequences. Quantitatively more RNA was synthesized from chromatin of 24 h estrogen-treated (E 24) chicks than from that of untreated chicks. In either case the size of transcribed RNA ranged from 5S to larger than 28S and most was between 5S and 18S. When a fraction enriched in estrogen receptor (E-Rec) complex was added to chromatin from untreated chicks, a dramatic shift of the RNA transcribed into heavier regions occurred. Analysis of RNA transcripts by hybridization to cDNAvit showed an equal number of sequences transcribed from E 24 chromatin and control; however, 13 times more specific sequences were transcribed in the presence of E-Rec complex. The results indicate the E-Rec complex exerts a regulatory function in the specific transcription of the vitellogenin gene.

Storage proteins in Zea mays (L.): interrelationship of albumins, globulins and zeins in the opaque-2 mutation.

Mature endosperms from opaque-2 mutant seeds with various genetic backgrounds (B 37, W 64A, R 802A and Oh 43) contained twice as much globulin than the corresponding normals, reduced amounts of zein and an increased amount of albumin. The latter is caused by a diminished disappearance of albumins in mutant endosperms during the final phases of development as compared to normal endosperms. Albumins from the opaque-2 endosperms appeared on gel electrophoresis as a similar but heterogenous polypeptide population comparable to that of normal endosperms except that in all opaque-2 forms a 70-kDa polypeptide was increased. Similarly, one to three specific globulin polypeptides (47 kDa, 52 kDa and 58 kDa) were, depending on the genotype, increased in the mutant lines. The accumulation of albumins, globulins and zeins, studied in developing W 64A opaque-2 mutant and corresponding normal maize kernels from 10 days after pollination until maturity, demonstrated two-phasic accumulation patterns for all proteins in the mutant, the first phase extending to about 30 days post pollination and the second one from there on until maturity. The transition time-point from first to second phase was characterized in mutant endosperm by a sudden reduction in accumulating albumins (also seen in normal endosperm), by an enhanced accumulation of globulins and the cessation of further accumulation of zein. Preferential accumulation of certain globulins has been found in the mutant during the second phase of globulin formation. The increased accumulation of globulins in the opaque-2 mutant endosperm is considered a response to the arrest in zein synthesis.

Storage protein characteristics of proline-requiring mutants of Zea mays (L.).

Protein and amino acid composition of mature karnels from three allelic proline-requiring mutants in maize, pro 1-1, pro 1-2, and pro 1-3 were analyzed and compared to kernels of the stock A 188 containing the wild type allele. The amount of free proline was specifically reduced in the embryos of all three mutants, while in the endosperm such a reduction was only found for pro 1-2 and pro 1-3 Accumulation of the proline-rich zeins was strongly reduced in the mutants, but in contrast to opaque-2 the reduction affected all major zein polypeptides to the same extent, possibly as a consequence of the defective proline metabolism. Albumins and globulins as well as free amino acids were more abundant in the endosperms of the mutants than in the wild type. Analysis of the albumins and globulins by SDS-PAGE revealed specific increases as well as reductions of certain polypeptides in the endosperms and embryos of the mutants.

Lysine biosynthesis and utilization during seed development of normal and opaque-2 Zea mays L.

This investigation deals with the metabolism of the amino acids from the aspartate family in an attempt to correlate the high free lysine content and the increase of lysine-containing storage proteins in mature opaque-2 mutant seeds. During seed development the changing levels of free aspartate, lysine, methionine, and threonine in endosperm as well as in embryos followed a bi-phasic pattern characterized by a minimum at 35 d post pollination. Up to that time, no striking differences were observed in any of these amino acids between normal and opaque-2 mutant tissues. However, during the second phase, increased levels of these amino acids in mutant endosperm- and to a certain extent in mutant embryos -indicate a feedback deregulation of one or several of the enzymes involved in the biosynthesis of amino acids originating from aspartate. The increases in these amino acids correlated with the demonstrated increased protein synthesis consisting mainly of endosperm and embryo globulins. Diaminopimelate decarboxylase (EC 4.1.1.20) activity was found to be equal in normal and mutant endosperm until 35 d post pollination, whereafter the activity declined sharply and appeared instead in embryos, especially in those of the mutant. Since lysine did not accumulate to any extent in mutant embryos, it is concluded that it was transferred to the endosperm, possibly for globulin synthesis. The data indicate that the embryo may play a specific role in controlling tissue levels of amino acids and thus be involved in the regulation of protein synthesis.

Stimulation of RNA polymerase I and II activities by 17 beta -estradiol receptor on chick liver chromatin.

The endogenous transcriptional capacity (RNA polymerase I and II activity) of liver chromatin from chicks treated with 17 beta-estradiol for 24 h (E 24) was double that of the controls. E 24 chromatin contained estradiol receptor activity while control chromatin did not. Its presence suggested an implication in the enhanced activities of RNA polymerases of E 24 chromatin. When semi-purified estradiol receptor was added to control chromatin, the endogenous transcriptional capacity of this chromatin was greatly increased. Studies with alpha-amanitin showed that both RNA polymerase I and II were stimulated by the estradiol receptor. This stimulation was observed as long as homology of the system was maintained. Solubilized homologous RNA polymerases were stimulated much less by the hormone complex in the presence of heterologous DNA than with homologous chromatin. Prokaryotic RNA polymerase could not be stimulated by chick liver estradiol receptor in the presence of heterologous DNA.

Multiple forms of DNA-dependent RNA polymerase I from immature chick liver. Selective effect of 17beta-estradiol.

1. We found that a single injection of 17beta-estradiol into immature chicks resulted within 24 h in a 2-fold increase of the transcripitional capacity of liver nuclei which involved both endogenous RNA polymerase I and II actvities. Similarly, RNA polymerase activity I of purified nucleoli was also doubled. 2. During purification of RNA polymerase I from controls and treated chicks the difference in activity was almost completely lost. 3. RNA polymerase I could be resolved into two forms, IA and IB, on CM-Spehadex. Form IA increased and form IB decreased after estrogen treatment while the sum of the two remained constant. Form IA sedimented more slowly than form IB in glycerol gradients and was predominant in purified nucleoli. 4. These observations suggest that there may be an equilibrium between the two forms of RNA polymerase I which may change in order to produce the translational shift-up which occurs after estrogen treatment.

Metabolism of Proline, Glutamate, and Ornithine in Proline Mutant Root Tips of Zea mays (L.).

In excised pro(1-1) mutant and corresponding normal type roots of Zea mays L. the uptake and interconversion of [(14)C]proline, [(14)C]glutamic acid, [(14)C]glutamine, and [(14)C]ornithine and their utilization for protein synthesis was measured with the intention of finding an explanation for the proline requirement of the mutant. Uptake of these four amino acids, with the exception of proline, was the same in mutant and normal roots, but utilization differed. Higher than normal utilization rates for proline and glutamic acid were noted in mutant roots leading to increased CO(2) production, free amino acid interconversion, and protein synthesis. Proline was synthesized from either glutamic acid (or glutamine) or ornithine in both mutant and normal roots; it did not accumulate but rather was used for protein synthesis. Ornithine was not a good precursor for proline in either system, but was preferentially converted to arginine and glutamine, particularly in mutant roots. The pro(1-1) mutant was thus not deficient in its ability to make proline. Based on these findings, and on the fact that ornithine, arginine, glutamic acid and aspartic acid are elevated as free amino acids in mutant roots, it is suggested that in the pro(1-1) mutant proline catabolism prevails over proline synthesis.

Characterization of chick liver chromatin and analysis of its in vitro transcription products.

Carefully controlled preparation of chromatin from purified chick liver nuclei yielded over 50% native chromatin as shown by the analysis of the nucleosome pattern after micrococcal nuclease digestion. The size of DNA in this chromatin as analyzed on alkaline sucrose gradients varied from 10S to 19S, the majority being 14S. All endogenous RNA polymerases were represented in the chromatin preparation although to different extents: RNA polymerase I was the most and RNA polymerase II the least abundant. Initiation studies showed that endogenous RNA polymerase II was capable of initiating RNA chains during 5 min. Saturation of chromatin with purified homologous RNA polymerase II increased initiation to 10 min. The addition of heparin caused the RNA transcribed to be larger in size and of increased yield. Chromatin transcription with added purified RNA polymerase II in the presence of heparin produced RNA as large as 32S. A chromatin preparation of this kind would therefore be suitable to transcribe any eukaryotic gene invitro provided additional homologous RNA polymerase II is used.Images

Pathway of urokinase-type plasminogen activator induction in the T47D and LLC-PK1 cell lines.

Induction of urokinase-type plasminogen activator (uPA) in response to either reagents activating cAMP-dependent protein kinase (cAMP-PK) or the calcium ion phospholipid-dependent kinase (C-kinase) was compared in the LLC-PK1 and T47D cell lines. The two cell lines exhibited quantitatively different responses to calcitonin, to the phosphodiesterase inhibitor isobutylmethylxanthine, and to the adenylate cyclase activator forskolin. Both showed activation of cAMP-PK in response to all these reagents, with T47D cells displaying a greater extent of activation. T47D cells, however, failed to produce uPA in response to calcitonin, forskolin, or the cAMP analog 8-bromo-cAMP, whereas LLC-PK1 cells produced high levels of uPA in response to all these agents. Both cell lines responded to phorbol esters in terms of uPA induction, though to differing extents. Phorbol myristate acetate (PMA) was shown conclusively not to activate cAMP-PK in either cell line, even at concentrations 10-fold higher than those promoting maximal uPA induction. It was concluded that phorbol ester-mediated induction of uPA does not involve cAMP or cAMP-PK activation. These results are discussed in relation to proposed models concerning the role of cAMP-PK in uPA induction.

Expression of zein in long term endosperm cultures of maize.

Continuous cultures, established 10 days after pollination from endosperms of inbred A636 Zea mays (L.) were extracted 21 months later with aqueous ethanol. The solubilized proteins were analyzed by poly-acrylamide-sodium dodecyl sulfate gel electrophoresis. Two protein bands co-migrated with zein, the major storage protein of maize. Immunoblotting of the gel followed by incubation of the immobilized proteins with anti-zein IgG provided evidence that the polypeptides were in fact zein. Electron microscopic studies showed that the cultures contained cells with protein bodies as found in developing endosperms. The protein bodies could be isolated from the cultures and were shown to contain zein. We conclude that the long term cultures described here synthesize zein and deposit it in the form of protein bodies of the type found in developing endosperms. Thus, certain endosperm characteristics and the production of tissue-specific proteins are retained in prolonged culture.

Effects of amiprophosmethyl on massive and limited calcium loading of maize mitochondria.

In maize mitochondria the effect of the herbicide amiprophosmethyl was studied on massive and limited Ca2+-loading and on mitochondrial energy transduction. Under massive Ca2+-loading conditions amiprophosmethyl inhibited the Ca2+ transport system directly without a significant effect on the respiratory chain, the membrane potential or Ca2+ efflux. Under limited Ca2+-loading conditions an increased Ca2+ efflux was noted, which could be partially responsible for the inhibition of the net Ca2+ accumulation.

Staurosporine stimulates expression of the urokinase-type (u-PA) plasminogen activator in LLC-PK1 cells.

In LLC-PK1 porcine epithelial cells, the urokinase-type plasminogen activator (u-PA) mRNA and protein can be induced either by stimulation of the protein kinase C (PKC) pathway using a tumor promoter (PMA) or by stimulation of the protein kinase A (PKA) pathway with calcitonin (SCT). By contrast, addition of 10(-7) M staurosporine, an inhibitor of PKC, to LLC-PK1 cells also stimulated urokinase production. In contrast to the in vitro situation (where staurosporine inhibited PKC activity), in the cell-culture system the microbial agent caused an early translocation of PKC and inhibited PKA. Addition of staurosporine together with PMA or with SCT further increased urokinase mRNA and protein synthesis. Maximal stimulation was obtained when all 3 agents were added together. We thus assume that in LLC-PK1 cells the PKA and PKC signal-transferring pathways can function independently.