THE EFFECT OF DENERVATION ON INCORPORATION OF 32P AND [3H]GLYCEROL BY THE MUSCLE MEMBRANE

Abstract— The incorporation in vivo of ³²P₁ was significantly increased in all glycerophosphatide of preparations of denervated muscle membrane in frogs. There was no increase in incorporation of ³²P₁ into sphingomyelin. Disuse induced by tenotomy did not significantly increase incorporation of ³²P₁ into phospholipids of the muscle membrane. The phospholipid content of muscle membranes remained unchanged as a result of denervation or tenotomy. Denervation produced an increase in the incorporation of [2-³H]glycerol into all glycerophosphatides in parallel with the increase in ³²P₁ incorporation. Although the stimulated incorporation of ³²P₁ was increased in the regions of the muscle membrane rich in endplates, the most marked effect was in the endplate-poor region where activity in phosphatidylserine was most markedly increased.     

Pilus biogenesis and epithelial cell adherence of Neisseria gonorrhoeae pilC double knock-out mutants

The phase-variable PilC proteins of pathogenic Neisseria species have recently been implicated in both assembly and cellular adherence functions of the type 4 pili of these pathogens. We describe here the cloning of full-length pilC1 and pilC2 genes and the complete sequencing of the pilC2 gene of Neisseria gonorrhoeae MS11. Sequential inactivation of both genes by gene replacement in piliated (P⁺) variants of N. gonorrhoeae MS11 led initially to a non-piliated (P⁻) phenotype; however, spontaneous P⁺ variants could be derived from some pilC1,2 double mutants which produced morphologically intact pili. Purified pili from pilC1,2 mutants revealed no detectable PilC protein. Instead, a novel protein about 70 kDa in size appeared in the pili preparations of P⁺ mutants; this protein exhibited no immunological cross-reactivity with PilC1 or PilC2. We propose that this novel factor replaces the function of PilC in pilus biogenesis. Using isogenic N. gonorrhoeae strains which produce identical PilE (pilin) proteins we demonstrate that pili associated with the 70 kDa protein do not confer gonococcal adherence to human epithelial cells, in contrast to pili assembled in the presence of PilC1 or PilC2.     

EFFECTS OF DENERVATION ON THE INCORPORATION OF 32P INTO PHOSPHATIDYL INOSITOL AND OTHER PHOSPHOLIPIDS OF RAT DIAPHRAGM

Abstract— At 24 h after denervation of the rat hemidiaphragm, incorporation of ³²P into phosphatidyl inositol was depressed relative to incorporation of ³²P into phosphatidyl choline (measured 75 min after injection of the isotope intraperitoneally). The ratio of the specific radioactivity of phosphatidyl choline to the specific radioactivity of P_i was unaffected by denervation which implies that denervation had depressed incorporation of isotope into phospatidyl inositol. Denervation did not cause a measurable change in the pool size of phosphatidyl inositol relative to that of phosphatidyl choline. The effect of denervation on incorporation of ³²P into phosphatidyl inositol was not entirely a direct consequence of the cessation of ACh release at the motor end-plate since the effect was clearly manifest in strips of muscle not containing motor end-plates, but the magnitude of the denervation effect was slightly greater in the strips of denervated hemidiaphragm which contained motor end-plates.     

Links between methanotroph community composition and CH4 oxidation in a pine forest soil

The main gap in our knowledge about what determines the rate of CH₄ oxidation in forest soils is the biology of the microorganisms involved, the identity of which remains unclear. In this study, we used stable-isotope probing (SIP) following ¹³CH₄ incorporation into phospholipid fatty acids (PLFAs) and DNA/RNA, and sequencing of methane mono-oxygenase ( pmoA ) genes, to identify the influence of variation in community composition on CH₄ oxidation rates. The rates of ¹³C incorporation into PLFAs differed between horizons, with low ¹³C incorporation in the organic soil and relatively high ¹³C incorporation into the two mineral horizons. The microbial community composition of the methanotrophs incorporating the ¹³C label also differed between horizons, and statistical analyses suggested that the methanotroph community composition was a major cause of variation in CH₄ oxidation rates. Both PLFA and pmoA -based data indicated that CH₄ oxidizers in this soil belong to the uncultivated 'upland soil cluster α'. CH₄ oxidation potential exhibited the opposite pattern to ¹³C incorporation, suggesting that CH₄ oxidation potential assays may correlate poorly with in situ oxidation rates. The DNA/RNA-SIP assay was not successful, most likely due to insufficient ¹³C-incorporation into DNA/RNA. The limitations of the technique are briefly discussed.     

Cell cycle related [3H]thymidine uptake and its significance for the incorporation into DNA

Abstract. Cellular uptake of [³H]thymidine ([³H]TdR) and incorporation into DNA of Ehrlich ascites tumour cells were studied in relation to the cell cycle by measuring the activity in the acid-soluble and insoluble parts of the cell material. Cells were synchronized at various stages of the cell cycle using centrifugal elutriation. The degree of synchrony of the various cell fractions was measured by flow-cytofluorometric DNA analysis. From the cellular uptake, the TdR triphosphate (dTTP) concentration of a mean cell in an unseparated cell population was calculated to be 20 × 10^-18 mol/cell. The pool activity of G₁ cells was unmeasurable but rose to maximum values at the border of the G₁-S phase. It decreased again during G₂. The [³H]TdR incorporation into DNA was low during early S phase, reached a maximum value at two-thirds of the S phase and decreased again during late S phase. These changes in DNA synthesis were not due to changes in the dTTP pool being a limiting factor. During maximum DNA synthesis, 10%× min^-1 of the dTTP pool was utilized, at which time the pool size also decreased by about 30%. Changes in pool size during the cell cycle have to be taken into account when the results of incorporation of radioactive TdR into DNA are discussed.     

NUCLEAR CHROMATIN PROTEINS FROM RABBIT CEREBRUM, CEREBELLUM AND LIVER: SYNTHESIS AND PHOSPHORYLATION

Abstract— In order to investigate synthesis and phosphorylation of the various fractions of nuclear proteins. [³H]leucine and [³²P] phosphate incorporation were studied with tissue slices in vitro. Cerebral cortex and cerebellum were used to delineate the similarity and dissimilarity within CNS, and liver was taken to compare the extraneural organ. There were significant differences in [³H]leucine incorporation into nuclear proteins among those tissue sources examined, while [³²P]phosphate incorporation showed very similar results among them. Although the acidic chromatin protein demonstrated high activity in each tissue source for both synthesis and phosphorylation, 0.14M-NaCl soluble protein showed the activity as high as or even higher than the acidic chromatin protein. Both [³H]leucine incorporation and [³²P]phosphate incorporation were relatively low in histone. When the acidic chromatin protein was further fractionated with SDS-acrylamide gel electrophoresis, significant difference was found between CNS tissue and liver for synthesis and phosphorylation. However, considerable difference was also observed even between cerebral cortex and cerebellum. The present investigation demonstrated complicity and diversity of nuclear chromatin proteins in different organs, not only for their protein constituents but also for their synthesis and phosphorylation.     

Thymidine incorporation into lake water bacterioplankton and pure cultures of chemolithotrophic (CO, H2) and methanotrophic bacteria

Abstract Incorporation of [³H]methyl thymidine into bacterial DNA was measured using samples of bacterioplankton from Lake Constance and pure cultures of CO, H₂ and CH₄-oxidizing bacteria. Thymidine was incorporated by Pseudomonas carboxydovorans, Paracoccus denitrificans, Methylosinus trichosporium, Methylomonas agile , and by various chemolithotropic or methylotrophic isolates from Lake Constance. Thymidine incorporation by bacterial cultures was stimulated by increasing concentrations of CO or H₂. Increased CH₄ concentrations stimulated thymidine incorporation by Ms. trichosporium only if the cells had been starved. In contrast to bacterial cultures, thymidine incorporation by bacterioplankton samples was not stimulated by increasing     

Probable Contribution of Protein Phosphorylation by Protein Kinase C to Spicule Formation in Sea Urchin Embryos

The formation of spicules and development of pluteus arms in sea urchin embryos were strongly blocked by H-7 (1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride) but were not affected by HA1004 ( N -(2-guanidinoethyl)-5-isoquinolinesulfonamide hydrochloride). Archenteron formation occurred normally in the presence of these compounds. Late gastrulae (28 hr after fertilization) were exposed to ³²Pi for 30 min at 20°C, and then dissociated and their primary mesenchyme cells with spicules, embryo-wall cells and archenteron cells were separated. Then, the radioactivities in the protein fractions of these separated cells were measured. Results showed that culture of embryos with H-7 strongly inhibited ³²p incorporation into proteins in primary mesenchyme cells but caused little inhibition of its incorporations in embryo-wall cells and archenteron cells. The effective concentrations of H-7 for inhibition of ³²p incorporation were within the range that blocked spicule formation and growth of pluteus arms in embryos. HA1004 only slightly inhibited ³²p incorporation into protein in mesenchyme cells, embryo-wall cells and archenteron cells of embryos exposed to ³²Pi. Protein kinase C activity was detectable only in isolated primary mesenchyme cells associated with spicule structures. These suggest that phosphorylation of proteins by protein kinase C contributes to the formation of spicule structures.     

Alterations of Lipid Metabolism in Response to Nerve Growth Factor

Abstract: In response to nerve growth factor (NGF), clonal pheochromocytoma cells flatten and extend neurites capable of making functional synapses. Although no significant changes in overall phospholipid composition occur in the presence of NGF, there is increased incorporation of ³²PO₄ into phosphatidylinositol and phosphatidic acid within 10 min after the addition of NGF. NGF stimulates the incorporation of ³²PO₄ into other lipids, such as phosphatidylcholine, to a lesser extent. The kinetics of the NGF-induced phosphatidylinositol responses are different when the cells are in suspension from when they are attached to culture dishes. These changes in phospholipid metabolism are discussed with respect to their role in NGF-induced nerve differentiation.     

NOREPINEPHRINE INCREASES THE [32P]LABELLING OF A SPECIFIC PHOSPHOLIPID FRACTION OF POST-SYNAPTIC PINEAL MEMBRANES

Abstract— Cultured pineal glands incorporated ³²P into membrane phospholipids. Treatment of cultured glands with norepinephrine, which is known to stimulate membrane- bound pineal adenyl cyclase and to increase the production and secretion of melatonin, stimulated the incorporation of ³²P into a phospholipid fraction of membranes and particulates containing phosphatidyl serine and phosphatidyl inositol. The labelling of other phospholipid fractions and the total ³²P in the gland were not changed by norepinephrine treatment. Experiments with chronically-denervated pineal glands indicated that the effect of norepinephrine on the [³²P]labelling of phospholipids occurred at a postsynaptic site. When norepinephrine-stimulated secretion of melatonin was partially inhibited by p -chlorophenylalanine (a compound which blocks the synthesis of melatonin precursors), the norepinephrine-stimulated labelling of phospholipids was still observed. Conversely, when melatonin secretion was stimulated in the absence of norepinephrine by treatment with the immediate precursor of melatonin, N -acetylserotonin, a stimulation of ³²P- labelling of phospholipids did not occur. These observations suggest that the increased [³²P]- labelling of a phospholipid fraction caused by the norepinephrine treatment is not related to the secretion of melatonin. This effect on phospholipids may be associated with the interaction of norepinephrine with a membrane-bound postsynaptic receptor. Stimulation by norepinephrine of [³²P]-incorporation into phospholipids has not been previously reported to occur in a tissue in which cholinergic fibres are absent.     

The vestigial teeth of Miniopterus schreibersii natalensis (Mammalia: Chiroptera)

Miniopterus schreibersii nufalensis has a vestigial tooth on both sides of the upper jaw between the canine and following premolar. This tooth is regarded as permanent premolar P² in vestigial form.     

Receptor-mediated increases in phosphatidylinositol turnover in neuron-like cell lines

Abstract: Muscarinic receptors found in the N IE-115 mouse neuroblastoma cell line were tested for their ability to mediate stimulation of phosphatidylinositol (PI) turnover. This study was facilitated by the development of a new solvent system (acetone: butanol: acetic acid: water, 5: 5: 1: 1) for the rapid and consistent separation of PI by one-dimensional thin-layer chromatography. Cholinergic stimulation caused as much as a 680% increase in the incorporation of ³²P into PI. Enhanced incorporation of ³²P into PI could be measured as early as 4 min after stimulation began. By 20 min, the rate of incorporation by stimulated cells had decreased to that of unstimulated cells, indicating desensitization. The magnitude of the response was dependent on the extent of receptor occupancy and the response elicited by a saturating dose of carbamylcholine was blocked completely by 10⁻⁷ M at-ropine, a specific muscarinic antagonist. Chronic stimulation, known to cause a loss of receptor binding sites, led to a 90% decrease in the maximum response even after a 40-min withdrawal period. Replacement of Na⁺ ions in the medium with choline or K⁺ severely impaired the ability of the cells to incorporate added ³²P into PI (90 and 50%, respectively). Removal of the putative second messenger Ca²⁺ for short periods of time by the addition of excess EGTA did not alter either basal or muscarinic-stimulated PI turnover.     

AUTORADIOGRAPHIC DETECTION OF DNA POLYMERASE CONTAINING NUCLEI IN SARCOMA 180 ASCITES CELLS

A technique has been developed allowing the autoradiographic detection of incorporation of ³H-thymidine-5'-triphosphate (³H-TTP) into nuclear DNA of smears of Sarcoma-180 (S-180) mouse ascites tumors under the direction of the cell's own nuclear DNA polymerase. Dried smears are dipped into an agar solution, which strips cytoplasm from the nuclei, and are then air dried and incubated with a buffered mixture containing four nucleotide triphosphates (one labeled), Mg⁺⁺, and Ficoll, with the cell's own DNA acting as primer. The incorporation of ³H-TTP into the nuclei, like the cell free DNA polymerase assay, is largely dependent on the presence of all four nucleotide triphosphates and Mg⁺⁺ and produces a product which is DNase sensitive and RNase resistant.
DNA polymerase activity, as studied in a cell free assay, decreases with tumor age. This correlates well with a decreasing ³H-TTP labeling index in autoradiographs of aging tumors. The ³H-TTP labeling index has also been shown to exceed but parallel the in vivo ³H-thymidine (³H-TdR) pulse labeling index for all tumor ages examined.
In at least some cell systems DNA polymerase seems characteristic of cells in cycle. The autoradiographic detection of nuclei containing the enzyme offers a new tool for the study of tumor cytokinetics.     

STUDIES ON LIVER REGENERATION: I. 131IODODEOXYURIDINE AS A PRECURSOR of DNA IN NORMAL and REGENERATING RAT LIVER

Abstract. ¹³¹Iododeoxyuridine (¹³¹IDU) was injected into normal and partially hepatectomized rats, and the specificity of incorporation of this thymidine analogue into liver DNA was determined 2, 24 and 48 hr following intramuscular injection. At 2 and 24 hr after ¹³¹DU injection, a major proportion of radioactivity in the liver was in the acid-soluble fraction, whereas 48 hr after injection the label in the acid-soluble fraction had decreased considerably. In liver obtained 2 hr after injection of ¹³¹IDU, only 1.8–16.6% of the total radioactivity were in DNA. If, however, the tissue was subjected to formalin fixation, the acid-soluble label was extracted selectively, and of the remaining radioactivity 64–88% was in DNA. Therefore, the radioactivity that is not extracted by formalin may be used as a measure of DNA synthesis at the time of injection of ¹³¹IDU, thus obviating time-consuming biochemical fractionation procedures.     

THE EFFECT OF DIABETES, INSULIN AND WALLERIAN DEGENERATION ON LEUCINE METABOLISM OF ISOLATED RAT SCIATIC NERVES

Abstract– ¹⁴CO₂ production and ¹⁴C incorporation into proteins was studied in isolated rat sciatic nerves during incubation with 0.1 mM-[1-¹⁴C]leucine. Rats were made diabetic with streptozotocin. Nerves from diabetic rats incubated with glucose oxidized more [¹⁴C]leucine than controls. This difference was abolished in the presence of insulin (1 mU/ml). The effects of diabetes and insulin on leucine oxidation could not be demonstrated in the absence of glucose. Insulin stimulated the incorporation of [¹⁴C] from leucine into proteins by nerves from controls and diabetic rats.
Nerves undergoing Wallerian degeneration showed a marked increase in DNA content and stimulated incorporation of [¹⁴C]leucine into proteins. ¹⁴CO₂ production from leucine proceeded at 75% of the rate observed in intact nerves. Neither insulin nor diabetes affected leucine metabolism in degenerating nerves.
Neither the extracellular space nor the concentration of free amino acids were significantly different in nerves obtained from control and diabetic rats, except for lower glutamine content in the latter.
In vitro leucine metabolism of nerves is affected by diabetes, insulin and the integrity of the axon. The Schwann cell is suggested as a possible site of the observed changes in leucine metabolism.     

Action of cortisol on the proliferation of rainbow trout fibroblasts

Abstract The effect of cortisol on the proliferation of the rainbow trout fibroblast cell line, RTG-2, was examined in synchronous and asynchronous cultures. When the transition from G₁ to S was synchronized by restoring serum to serum-deprived cultures, the addition of cortisol at the time of serum restoration delayed the entry of cells into S phase. However, if cortisol was added 24 h after serum restoration, at the G₁/S transition point, the subsequent peak of DNA synthesis was unaffected. In asynchronous cultures cortisol inhibited [³H]-thymidine and [³H]-uridine but not [³H]-leucine incorporation into acid-insoluble material. If the exogenous nucleoside concentration was raised, [³H]-thymidine but not [³H]-uridine incorporation continued to be inhibited by cortisol. This suggested that cortisol's effect on [³H]-thymidine incorporation reflected a change in entry into S phase and not just on thymidine uptake and metabolism. Cortisol inhibited the proliferation of RTG-2 in asynchronous cultures. At 1000 ng/ml of cortisol a reduction in cell number became apparent before the RTG-2 cultures were confluent, whereas at 100 ng/ml the reduction only became evident in confluent cultures. The synthetic antiglucocorticoid, RU 486, which acts at the level of the corticosteroid receptor, blocked the growth inhibition by cortisol. These results suggest that cortisol regulates rainbow trout fibroblast proliferation via the corticosteroid receptor and that the G₁/S transition is one point at which this regulation occurs.     

Some Effects of Pentamidine Di-isethionate on Crithidia fasciculata

SYNOPSIS. Growth-inhibitory concentrations of pentamidine inhibit to a similar extent net synthesis of DNA, RNA, protein and phospholipid by washed cell suspensions of Crithidia fasciculata. The incorporation of ³H-thymidine into DNA, ¹⁴C-adenine into DNA and RNA and ¹⁴C-lysine into protein is similarly inhibited. The same concentrations of drug have little or no effect on viability, motility, 1-C metabolism, respiration or K⁺ content of organisms, altho they do cause increased amounts of intracellular ATP. Lysine uptake (in the absence of arginine) is, however, inhibited. At higher concentrations of drug respiration is inhibited and organisms lose K⁺. The mode of action of pentamidine is considered in the light of these observations and a mechanism of uptake of pentamidine into organisms is suggested.     

Reinitiation of DNA Synthesis in Postmitotic Nuclei of Myotubes by Virus-Mediated Fusion with Embryonic Fibroblasts

Myotubes, whose nuclei have stopped DNA synthesis were fused with replicative embryonic fibroblasts. In heterokaryons the postmitotic muscle nuclei resumed DNA synthesis. Incorporation of radioactive thymidine into muscle, and also into fibroblast nuclei was dependent upon the time elapsed between virus-mediated fusion and administration of radioactive thymidine. Whereas incorporation into fibroblast nuclei diminished with time, there was an early increase of labelling into muscle nuclei followed by a decrease of incorporation of ³H thymidine. DNA synthesis was also dependent upon the ratio of noncycling (muscle) to cycling (fibroblast) nuclei. There was a greater incorporation of ³H thymidine into muscle and fibroblast nuclei in myotubes containing larger numbers of fibroblast nuclei. A model is discussed for the control of DNA synthesis in polykaryocytes derived from fusion of cycling and noncycling cells.     

Effects of dichloroisopropylarterenol on respiration, nucleotide ∼ P and phospholipids of slices of cerebral cortex

In a Recent report Hokin (1969) showed that norepinephrine stimulated the incorporation of ³²P into phosphatidic acid (PA) of slices of cerebral and cerebellar cortex and into phosphaditylinositol (PI) of slices of cerebral cortex. In the course of our experiments on agents affecting the metabolism of phospholipids, the effects of epinephrine and its antagonist, dichloroisopropylarterenol (DCI), on the labelling of energy-rich nucleotides and phospholipids of cerebral tissue have been measured. Epinephrine had no significant effect but DCI stimulated the incorporation of ³²P into PA by 165 per cent and into PI by 90 per cent.