Protein Synthetic Activities during Spermiogenesis in the Sea Urchin: An high Resolution Autoradiographic Study of 3H-Leucine Incorporation

Protein synthetic activity has been studied during spermiogenesis of Paracentrotus lividus by high-resolution autoradiography using ³H-leucine as a labeled precursor. Under the adopted experimental conditions ³H-leucine is incorporated during the whole spermiogenesis period. The early spermatid is the most active stage and it shows labeling over the nucleus, the cytosol and the mitochondria. Nuclear ³H-leucine incorporation progressively decreases as spermiogenesis proceeds. Cytosol labeling shows similar values at early and intermediate spermatid and it undergoes a considerable decreases at late spermatid. Mitochondrial grain density increases from early to intermediate spermatid and it remains almost constant at late spermatid.
Our results are compared with the data reported for other animal groups and possible functions of the observed protein synthesis are discussed.     

Cytokinin-induced switch in development in excised cotyledons of radiata pine cultured in vitro

Cotyledons of Pinus radiata D. Don were cultured under shoot-forming (plus cytokinin) and elongating (minus cytokinin) conditions. Using. autoradiographic and precursor incorporation techniques, the sites and rate of macromolecular synthesis were examined during the first five days in culture. Active incorporation of ³H-thymidine, ³ H-uridine and ³H-leucine occurred. In shoot-forming cotyledons the incorporation became preferentially located in the epidermal and sub-epidermal cell layers in contact with the medium. In elongating cotyledons, in contrast, incorporation was randomly distributed, and the amount of incorporation declined with time. Biochemically, differences in DNA, RNA and total protein synthetic patterns were observed. In elongating cotyledons the rates of RNA and protein synthesis were higher during the first 48 h than in shoot-forming tissues, after which the synthetic rates were similar. Two peaks of newly formed DNA were observed in both tissues. These findings indicate that the cytokinin-induced changes in developmental pathways began within 24 h in culture.     

Bacterial production measured by leucine and thymidine incorporation rates in French lakes

1. Production of heterotrophic bacterioplankton was estimated monthly by the tritiated thymidine and leucine incorporation methods during the draining and filling of the mesotrophic Lake Pareloup (over a 2.5-years sampling program).
2. Rates of ³H-leucine (leu) and ³H-thymidine (thy _DNA) incorporation generally paralleled each other but the ratio of leu/thy _DNA incorporation rates was higher for the draining period (34.5 mean) than during and after filling (11.5 mean).
3. After draining, the highest ratios were observed during periods of low temperature and low bacterial specific activity, while DNA labeling by ³H-thymidine was reduced. However, bacterial production estimates obtained by ³H-leucine (BPL) and ³H-thymidine (BPT) incorporation methods were generally well correlated and the average BPL/BPT ratio was equal to 0.78.
4. In addition, both methods were applied during a diel cycle in three lakes of different trophic status. An increase of leu/thy _DNA incorporation rates was noted from the oligotrophic to the eutrophic system. In the absence of Cyanobacteria, BPL and BPT values were quite concordant on average.
5. In situations of unbalanced growth, BPL and BPT values can diverge but when considered over a sufficient period of time they were found to be in agreement.     

Effects of retinoic acid on cell proliferation and cell differentiation in a rat tracheal epithelial cell line

Abstract. The effects of 3.3 times 10-⁷ M to 3.3 times 10^-5 M all- trans -retinoic acid (vitamin-A acid) on the total cell population dynamics of 165 S, a keratinizing epithelial cell line from carcinogen-exposed rat trachea, were studied. During the first 6 days of culture, cells accumulated on the dish in the presence of the vitamin to twice the density of controls. [³H]thymidine incorporation into DNA and percentage of [³H]thymidine-labelled cells in autoradiographs were stimulated in a dose-dependent fashion to a maximum of 25- and 34-fold, respectively. Exfoliation of cells from the cultures was also enhanced 2–3-fold, resulting in nearly twice the total number of cells (attached plus exfoliated) in the presence of the vitamin.
During 19 days of culture, retinoic acid maintained a higher level of [³H]thymidine incorporation and cell exfoliation in 165 S cells so that by day 19, total cell production was more than three times that seen in controls. At this time, vitamin-treated cultures showed a reduced cell saturation density compared to controls. The higher final cell density in the control cultures was a result of multilayering and papillary formation which did not occur in the presence of retinoic acid. The papillae in control cultures stained specifically with Rhodamine B or with the eosin and orange G components of the Papanicolaou method. A count of the number of eosin and orange G positive cells in the attached and exfoliated cell compartments showed an 8-fold reduction of keratinization in retinoic acid-exposed cultures. Our results show that retinoic acid is a growth stimulant in these cell cultures, causing increased cell proliferation and exfoliation accompanied by inhibition of keratinization.     

Diel rhythmicity in amino acid uptake by Prochlorococcus

The marine cyanobacterium Prochlorococcus , the most abundant phototrophic organism on Earth, numerically dominates the phytoplankton in nitrogen (N)-depleted oceanic gyres. Alongside inorganic N sources such as nitrite and ammonium, natural populations of this genus also acquire organic N, specifically amino acids. Here, we investigated using isotopic tracer and flow cytometric cell sorting techniques whether amino acid uptake by Prochlorococcus is subject to a diel rhythmicity, and if so, whether this was linked to a specific cell cycle stage. We observed, in contrast to diurnally similar methionine uptake rates by Synechococcus cells, obvious diurnal rhythms in methionine uptake by Prochlorococcus cells in the tropical Atlantic. These rhythms were confirmed using reproducible cyclostat experiments with a light-synchronized axenic Prochlorococcus (PCC9511 strain) culture and ³⁵S-methionine and ³H-leucine tracers. Cells acquired the tracers at lower rates around dawn and higher rates around dusk despite >10⁴ times higher concentration of ammonium in the medium, presumably because amino acids can be directly incorporated into protein. Leucine uptake rates by cells in the S+G₂ cell cycle stage were consistently 2.2 times higher than those of cells at the G₁ stage. Furthermore, S+G₂ cells upregulated amino acid uptake 3.5 times from dawn to dusk to boost protein synthesis prior to cell division. Because Prochlorococcus populations can account from 13% at midday to 42% at dusk of total microbial uptake of methionine and probably of other amino acids in N-depleted oceanic waters, this genus exerts diurnally variable, strong competitive pressure on other bacterioplankton populations.     

Gibberellic acid-stimulated and auxin-stimulated cell growth in relation to RNA synthesis, protein synthesis and development in the wheat coleoptile

Young excised coleoptiles from dark grown wheat have their cell growth promoted by gibberellic acid (GA₃), while sections from older coleoptiles have their cell growth promoted by auxin. The GA₃ response has a much longer lag period than that of auxin. Neither GA₃ nor auxin has any effect on ¹⁴C-leucine and ¹⁴C-uridine incorporation and uptake after 1 h, indicating that the lag in growth stimulation following GA₃ application is not associated with changes in protein or RNA synthesis. Following a 6 h incubation there are small increases in ¹⁴C-leucine and ¹⁴C-uridine incorporation in response to both GA₃ and auxin, and in the case of auxin this is associated with increased uptake. Studies on protein and RNA turnover using pulse-chase experiments have shown that both GA₃ and auxin have no effect on protein and RNA stability. There are, however, developmental changes in RNA and protein synthesis that should be considered in any explanation of the mechanism of action of these hormones on cell growth. Young GA₃-sensitive tissue has high rates of RNA synthesis and low protein and RNA turnover, while auxin-sensitive tissue has low rates of RNA synthesis, slightly higher rates of RNA turnover and much higher rates of protein turnover. The evidence overall favours more effective utilisation by GA₃ and auxin of a basal control level of RNA and protein synthesis and turnover in coleoptile tissue.     

Assessing amino acid uptake by phototrophic nanoflagellates in nonaxenic cultures using flow cytometric sorting

Biologically available concentrations of individual dissolved amino acids in the open ocean are generally <1 nM. Despite this, the microbial turnover of amino acids is usually measured in hours indicating high demand. It is thought that the majority of uptake is due to bacterioplankton, although protists, particularly phototrophic protists, are also expected to take up amino acids. In order to assess the ability of protists to compete with prokaryotes for amino acids at subnanomolar concentrations, we examined the direct uptake of ³H-leucine by phototrophic nanoflagellates (prasinophytes, pelagophytes and trebouxiophytes) and by associated bacteria using flow cytometric cell sorting. In contrast to ³H-leucine-assimilating bacterial copopulations, none of the six studied nanoflagellates showed measurable direct uptake of ³H-leucine, suggesting that the studied phototrophic protists were unable to utilize dissolved ³H-leucine at natural oceanic concentrations. More practically, the flow-sorting technique allowed rapid and unequivocal differentiation of organic nitrogen uptake between prokaryotic cells and eukaryotic cells in mixed microbial populations, reducing the need to establish and maintain axenic algal cultures.     

ON THE MECHANISM OF OUABAIN INHIBITION OF SYNAPTOSOME PROTEIN SYNTHESIS

Abstract— Ouabain (200μ m ) inhibited incorporation of radiolabelled leucine or glycine into the protein of neonatal synaptosome fractions but had minimal effect on preparations from adult rats. Leucine uptake into synaptosomes was rapid but not influenced by 200μ m -ouabain in contrast to ouabain inhibition of [¹⁴C]glycine and [¹⁴C]γ-aminobutyric acid uptake. Ouabain blocked the Na⁺ -dependent (stimulated) component of synaptosome fraction protein synthesis in the presence of 25m m -K⁺. Ouabain inhibition was not alleviated by addition of ADP or ATP. 100μ m -atractylate failed to influence [³H]leucine uptake or incorporation. Synergistic inhibition by ouabain was observed with the cycloheximide-sensitive component of protein synthesis and the chloramphenicol sensitive phase. Increasing the medium Ca²⁺ concentration stimulated protein synthesis and this stimulated component was inhibited by ouabain. Ouabain inhibition was associated with decreasing intraterminal K⁺ concentration and [K]_i was linearly related to the protein synthesis rate in control and ouabain treated preparations.     

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.     

Retinoic acid induced proteoglycan release and cartilage resorption in rat bone cultures are age dependent and inhibited by EDTA

Summary In explanted humeri of late fetal rats, retinoic acid was found to induce the release of proteoglycan followed by cartilage resorption. Tissue breakdown, which was demonstrated by losses of DNA, RNA, and protein, coincided with the appearance of necrotic cells. In control humeri chondrocytes were the main cell type, but in humeri treated for 4 days with retinoic acid the surviving cells were chondroblastlike. Sensitivity of proteoglycan release and tissue breakdown to retinoic acid decreased with age.The proteinase inhibitors cysteine, Trasylol, and soya and lima bean trypsin inhibitors did not antagonize the effects of retinoic acid. Phenylmethanesulfonyl fluoride suppressed cartilage resorption more effectively than proteoglycan release, while pepstatin merely suppressed cartilage resorption. The inhibition by EDTA of both the release of proteoglycan and cartilage resorption induced by retinoic acid was dose dependent. Zn²⁺ abolished these effects, whereas Mn²⁺ only relieved the release of proteoglycan induced by retinoic acid; this indicates that these two effects of retinoic acid are not necessarily linked.In view of our recent demonstration that the release of proteoglycan induced by retinoic acid requires RNA and protein synthesis, we suggest that the degradation of proteoglycans in response to retinoic acid is dependent upon continued synthesis of metalloproteinases.     

METABOLISM OF GLUTAMATE AND RELATED AMINO ACIDS IN THE 10-DAY-OLD MOUSE BRAIN: EXPERIMENTS WITH LABELLED ACETATE AND β-HYDROXYBUTYRATE

Abstract– The pattern of incorporation of [³H, 1-¹⁴C]- and [³H. 2-¹⁴C]acetate into glutamate and related amino acids was studied in the brain of 10-day-old mice. A comparison of these patterns with those obtained for the adult brain led to the suggestion that the glutamate pool labelled directly by acetate is a much larger fraction of the total glutamate pool in the 10-day-old brain than it is in the adult brain.
Some data on the pattern of labelling of brain amino acids by 3-hydroxybutyrate. glucose and acetate support the hypothesis that direct carboxylation of pyruvate is somewhat more active in the immature than in the mature brain.
Differences in the labelling patterns of free and protein-bound brain amino acids by acetate, do indicate that the free amino acid pool labelled by acetate is not the precursor pool for protein synthesis.     

Development of Synaptic Glycoproteins: Effect of Postnatal Age on the Synthesis and Concentration of Synaptic Membrane and Synaptic Junctional Fucosyl and Sialyl Glycoproteins

Abstract: Synaptic plasma membranes (SPM) and synaptic junctions (SJ) were isolated from the cortices of rats varying in age between 5 and 28 days. Gel electrophoresis of SPM and SJ indicated a marked increase in the concentration of the "PSD protein" (M. W. 52,000) with development. The biosynthesis of glycoproteins was measured following the intracranial injection of [³H]fucose or [³H] N '-acetylmannosamine. The incorporation of [³H]fucose into synaptic fractions decreased two- to threefold between 10 and 28 days whereas little change in the incorporation of [³H] N '-acetylmannosamine occurred over the same period. Gel electrophoretic analyses of labeled synaptic membranes indicated major increases in the relative incorporation of radiolabeled precursors into glycoproteins with apparent molecular weights of 74,000, 65,000, 50,000, and 40,000 with increasing age. Identification of fucosyl and sialyl glycoproteins following reaction with ¹²⁵I-fucose-binding protein or labeling of sialic acid with NaIO₄NaB[³H₄] demonstrated similar increases in the concentrations of these glycoproteins. Synaptic junctions contained three major glycoproteins with apparent molecular weights of 180,000, 130,000 and 110,000. The reaction of these glycoproteins with ¹²⁵I-fucose-binding protein increased one- to twofold between 10 and 28 days but little variation in their relative distribution or synthesis occurred over this period. The reaction of synaptic junctional glycoproteins GP 180 and GP 110 with ¹²⁵I-wheat germ ag-glutinin increased between 10 and 28 days. The results indicate that the molecular composition of the synapse continues to evolve after the initial synaptic contact has been formed.     

Quantitative Analysis of Protein Synthesis Altered by Estrogen in Cultured Xenopus Liver Parenchymal Cell

Using the primary culture system of male Xenopus laevis hepatocytes consisting of more than 95% parenchymal cells, the effect of estradiol-17 β (10⁻⁶M) on protein synthesis was quantitatively analyzed by ³H-leucine incorporation kinetics and the estimation of specific radioactivity of newly synthesized secretory protein. The cells in a well defined culture revealed high plating efficiency and very low DNA synthetic activity. The cultured cells could synthesize several secretory proteins containing serum albumin. The pattern of secreted proteins in SDS-polyacrylamide gel electrophoresis (SDS-PAGE) did not alter with culture time but secretion rate of protein increased for 7 days, starting on the third day following inoculation. Estradiol added to the culture media extensively induced the synthesis of yolk precursor protein vitellogenin which accounted for 40–50% of the overall secretory protein synthesis and 20–30% of the total protein synthesis on day 7 of estradiol treatment. Ultimately, the total protein synthesis and secretory protein synthesis were stimulated 1.2–1.3 fold and 2.0–2.2 fold, respectively, over those of the control cells cultured in the absence of estradiol. These results indicated that the stimulation of protein synthesis was largely due to vitellogenin production. Such an estradiol-dependent stimulation of protein synthesis was also detected in the low molecular weight protein(s). On the other hand, albumin synthesis was evidently reduced by estradiol. Thus, estradiol had two different effects on protein synthesis.
The results obtained in this study will be discussed in relation to the findings o in vivo experiments.     

Inhibition of Protein Synthesis and Amino Acid Transport by Crystal Violet in Trypanosoma cruzi

ABSTRACT. [³⁵S]methionine incorporation into proteins of either T. cruzi epimastigotes or trypomastigotes was drastically inhibited by low concentrations of crystal violet in a dose-dependent manner. This inhibition was not due to ATP depletion since cellular ATP levels did not change significantly after incubation of epimastigotes with 50 μM crystal violet for similar periods of time, and was unaffected by changes in the extracellular free calcium concentration. Although crystal violet was able to inhibit protein synthesis in a cell-free system from T. cruzi epimastigotes, half maximal inhibition was at 1 mM, a concentration three orders of magnitude higher than those that inhibited protein synthesis in intact cells. On the other hand, crystal violet was able to inhibit total [³⁵S]methionine uptake at similar concentrations to those that inhibited protein synthesis while addition of increasing concentrations of cold methionine to the incubation medium protected the cells against crystal violet inhibition. Crystal violet also inhibited total [³H]proline uptake thus indicating that it has a general inhibitory effect upon the transport of amino acids, and not specifically upon methionine. These results indicate that inhibition of protein synthesis by crystal violet is probably due to inhibition of amino acid uptake.     

A Novel Photoaffinity Ligand for Kainate Sites Labels Two Polypeptides with Molecular Mass 45 and 33.5 kDa in Chick Cerebellum

Abstract: The synthesis of (2 S ,3 S ,4 S )-4-[1-(4-azidobenzamidomethyl)ethenyl]-2-carboxy-3-pyrrolidineacetic acid (ABCPA) is described. This novel kainic acid analogue, bearing a photolabile functionality on the isopropenyl side chain, was proven to be a good inhibitor of [³H]CNQX and [³H]kainic acid binding on chick cerebellar membranes. [³H]ABCPA was photoaffinity cross-linked on the membrane fraction of chick cerebellum. Electrophoretic analysis with sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed two major radioactive bands with apparent molecular masses of 45 and 33.5 kDa. [³H]ABCPA incorporation in both bands was completely blocked by 2 m M CNQX. When photoaffinity labeling was performed in the presence of 2 m M kainic acid, incorporation of [³H]ABCPA was blocked by ∼70% in the 45-kDa band and by 18% in the 33.5-kDa band. Incorporation of radioactivity in both bands was blocked by ∼30% with 10 m M glutamate.     

The Effects of Insect Juvenile Hormone on the Growth of Some Protozoans in vitro. I. Crithidia sp.

SYNOPSIS. Experiments were designed to investigate the effects of insect juvenile hormone (JH) on the over-all growth and macromolecular synthesis of Crithidia sp. in vitro. Cells grown in the presence of 10⁻⁵M-10⁻³M JH showed a concentration-dependent inhibition of growth, which appeared to result from both a prolongation of generation time and a delay in the onset of logarithmic growth. Juvenile hormone (10⁻³M) inhibited the incorporation of [³H]thymidine, [³H]uridine and [³H] leucine into logarithmically growing cells by 50, 70 and 40% respectively. The incorporation of [³H]uridine into acid insoluble material could be stopped within 1 hr of application of the hormone (10⁻³M). The inhibitory effect was reversible in terms of cell numbers in subcultures of washed cells but an examination of the reversibility of RNA synthesis inhibition suggested that the resumption of RNA synthesis at an optimal level would require a lag period of at least 1–3 hr. It is suggested that JH may act by interfering with RNA synthesis either directly or indirectly by primarily acting at the level of the plasma membrane.     

Effects of Monensin on Assembly of Po Protein into Peripheral Nerve Myelin

Abstract: The ionophore monensin has been used in a variety of systems to block secretion of glycoproteins or assembly of glycoproteins into membranes. We examined the effects of monensin on assembly of the Po glycoprotein into PNS myelin, and compared this agent with the glycosylation inhibitor tunicamycin in our system. Sciatic nerves from 9-day-old rat pups were sliced and incubated in vitro . Electron microscopy of the Schwann cells in slices incubated with monensin revealed extensive swelling of the Golgi complex. Incubation with 10⁻⁷ M monensin inhibited total protein synthesis by about 20% and fucose incorporation into protein about 35%. Following isolation of myelin, proteins were separated by sodium dodecyl sulfate gel electrophoresis. Monensin inhibited the appearance of Po in myelin, while causing its accumulation in a denser membrane fraction. In addition, a slightly faster-migrating species of P_o labeled with both [³H]fucose and [¹⁴C]glycine was observed in all fractions. Assembly of basic proteins into myelin was not affected. Preincubation with 10 μg/ml tunicamycin for 30 min prior to incubation with [³H]fucose and [¹⁴C]glycine for 2 h resulted in a 65% decrease in [³H]fucose incorporation into P_o, and the appearance of a new [¹⁴C]glycine-labeled peak that migrated in the region of the 23K protein reported by Smith and Sternberger. [³H]Fucose incorporation was inhibited earlier, and to a greater extent, than protein synthesis. Our results show that processing of the P_o glycoprotein is sensitive to both monensin and tunicamycin, and that monensin partially blocks assembly of P_o into myelin.     

Effect of an exogenous auxin on maize tissues. Alteration of protein synthesis and phosphorylation

Pérez, L., Aguilar, R. and Sánchez-de-Jiménez, E. 1987. Effect of an exogenous auxin on maize tissues. Alteration of protein synthesis and phosphorylation. - Physiol. Plantarum 69: 517–522.
A synthetic auxin 2-(2-methyl-4-chloro)phenoxypropionic acid (MCPP), analogue of 2,4-D, alters maize ( Zea mays L. H-30) germination while inducing callus formation. The effect of this auxin on protein synthesis and phosphorylation of the embryonic tissues was explored. Total cytoplasmic proteins were analysed for ¹⁴C or ³²P incorporation into trichloroacetic acid precipitable material. MCPP significantly stimulated protein synthesis as well as protein phosphorylation. The protein synthesis pattern was highly altered in the presence of MCPP as analysed by two-dimensional gel electrophoresis. Analyses by Sephadex G-100 chromatography and by two-dimensional gel electrophoresis of phosphorylated proteins indicate that the effect of MCPP on protein phosphorylation was only quantitative.     

Amino acid incorporation by a natural population of Oscillatoria rubescens. A microautoradiographic study

Abstract The incorporation of a very low concentration (0.015 μM) of an [³H]amino acid mixture was measured for a natural population of Oscillatoria rubescens DC in samples from a eutrophic lake, Lake Nantua (France).
The kinetics of amino acid incorporation in the size fraction ⩾12 μ m showed that uptake was fast and that the maximum was reached after 4 h.
Microautoradiography demonstrated that Oscillatoria rubescens is able to utilize for protein synthesis an external pool of amino acids whose [³H] label becomes distributed generally throughout the cell.     

Incorporation of label from root-applied N6[8-14C]furfiuryIadenine into the guanine nucleotide fraction of pea bud ribonucleic acid

The pattern of incorporation of label into the nucleotides of axillary bud ribonucleic acid was investigated in Pisum sativum L. cv. Meteor following the application of N ⁶[8-^I4C]furfuryladenine or of [8-¹⁴C]adenine to the root system of decapitated plants and to cultured excised buds. When N ⁶[8-¹⁴C]furifaryladenine was applied to the root system label was confined to the guanine nucleotide moiety of the axillary bud ribonucleic acid; label from [8-¹⁴C]adenine was incorporated preferentially into adenine nucleotide in the molar ratio adenine nucleotide/guanine nucleotide = 3.23. When isolated buds were incubated in media containing [8-¹⁴C]adenine or N ⁶[8-¹⁴C]furfuryladenine, label was incorporated into both purine moieties of the ribonucleic acid. However, the relative incorporation into the guanine nucleotide fraction was considerably greater for N ⁶[8-^I4C]furfuryladenine (adenine nucleotide/guanine nucleotide = 2.23) than for [8-¹⁴C]adenine (ratio = 4.67).
It was concluded that the pattern of metabolism of adenine to guanine and its incorporation into the guanine nucleotide moiety of pea axillary bud ribonucleic acid, is influenced by the presence of a substitution in the N ⁶ position of the adenine base.