Precursor Pool Size Research Articles

Unique Expression Patterns of Cell Fate Molecules Delineate Sequential Stages of Dentate Gyrus Development

The dentate gyrus of the hippocampus is uniquely organized with a displaced proliferative zone that continues to generate dentate granule cells throughout life. We have analyzed the expression of Notch receptors, Notch ligands, and basic helix-loop-helix (bHLH) genes during dentate gyrus development to determine whether the need to maintain a pool of undifferentiated precursors is reflected in the patterns of expression of these genes. Many of these genes are expressed diffusely throughout the cortical neuroepithelium at embryonic days 16 and 17 in the rat, just preceding the migration of newly born granule cells and dentate precursor cells into the dentate anlage. However, at this time, Mash1, Math3, and Id3 expression are all concentrated in the area that specifically gives rise to granule cells and dentate precursor cells. Two days later, at the time of migration of the first granule cells and dentate precursor cells, cells expressing Mash1 are seen in the migratory route from the subventricular zone to the developing dentate gyrus. Newly born granule cells expressing NeuroD are also present in this migratory pathway. In the first postnatal week, precursor cells expressing Mash1 reside in the dentate hilus, and by the third postnatal week they have largely taken up their final position in the subgranular zone along the hilar side of the dentate granule cell layer. After terminal differentiation, granule cells born in the hilus or the subgranular zone begin to express NeuroD followed by NeuroD2. This study establishes that the expression patterns of bHLH mRNAs evolve during the formation of the dentate gyrus, and the precursor cells resident in the mature dentate gyrus share features with precursor cells found in development. Thus, many of the same mechanisms that are known to regulate cell fate and precursor pool size in other brain regions are likely to be operative in the dentate gyrus at all stages of development.

Near-Isogenic Lines of Maize Differing for Glycinebetaine

A series of near-isogenic glycinebetaine-containing and -deficient F8 pairs of Zea mays L. (maize) lines were developed. The pairs of lines differ for alternative alleles of a single locus; the wild-type allele conferring glycinebetaine accumulation is designated Bet1 and the mutant (recessive) allele is designated bet1. The near-isogenic lines were used to investigate whether glycinebetaine deficiency affects the pool size of the glycinebetaine precursor, choline, using a new method for glycinebetaine and choline determination: stable isotope dilution plasma desorption mass spectrometry. Glycinebetaine deficiency in maize was associated with a significant expansion of the free choline pool, but the difference in choline pool size was not equal to the difference in glycinebetaine pool size, suggesting that choline must down-regulate its own synthesis. Consistent with this, glycinebetaine deficiency was also associated with the accumulation of the choline precursor, serine. A randomly amplified polymorphic DNA marker was identified that detects the bet1 allele. In 62 F8 families tested the 10-mer primer 5'-GTCCTCGTAG produced a 1.2-kb polymerase chain reaction product only when DNA from Bet1/bet1 or bet1/bet1 lines was used as template. All 26 homozygous Bet1/Bet1 F8 families tested were null for this marker.

Effects of drying/rewetting stress on microbial auxin production and L-tryptophan catabolism in soils

The presence of tryptophan in soil and auxin production by indigenous soil microbes are considered to be important natural plant growth-promoting factors. In order to elucidate the natural regulation of microbial auxin synthesis, we treated different soils by an air drying/rewetting cycle and measured pool sizes of auxins, auxin precursors, and degradation products of tryptophan together with a range of respiration parameters. Potential (tryptophan addition) microbial production of indole-3-acetic acid (auxin) was predominant in the equilibrated fresh soils. Auxin production depended on the soil nutrient content, and the size and metabolic status of the microbial biomass. Immediately after rewetting, potential auxin production was low, whereas potential indole-3-ethanol and anthranilic acid production as well as basal respiration were transitionally enhanced. This was concurrent with proliferation ofr-strategist microbes. After the respiration flush, the natural tryptophan contents increased, indicating cell lysis, probably caused by a rise in protozoan grazing on ther-strategists. Auxin production was high in fresh and in re-equilibrating rewetted soils, probably due to nutritional limitations under stationary conditions. Hence, this high production was attributed to theK-strategist component of the soil microflora. The differences observed in the recovery of auxin production between the different rewetted soils suggest that original activities can become re-established rapidly when the indigenous microbial community is pre-adapted to the stress. We propose that the release of tryptophan, microbial auxin, and the shift towards indole-3-ethanol production function as stimulants for root development induced by environmental fluctuations.

Estimates of bacterial productivity in marine sediments and water from a temperate saltmarsh lagoon

Tritiated thymidine incorporation (TTI) into DNA was used to estimate bacterial productivity in sediment and water samples from two sites in Langebaan Lagoon, South Africa. Routine analysis of isotope dilution showed seasonal variations of approximately threefold in the thymidine precursor pool sizes for bacterial assemblages from each site. Dual label incorporation of [(3)H]-thymidine and (14)C-leucine into DNA and protein, respectively, showed that pelagic but not sediment assemblages were in a balanced state of growth during TTI. This is the first report of dual label measurements of bacterial production in sediments. Sediments supported bacterial productivities that exceeded those in the water column by factors from five- to 950-fold, whereas bacterial abundance supported by sediments exceeded that in the water column by more than 3 orders of magnitude. Estimates of bacterial productivities in sediments were coincident with levels of organic content in sediments, but not with bacterial abundance. Measurements of TTI activity for 5 different benthic microhabitats at one lagoon site showed highest activity associated with seagrass beds (2.11 ± 0.84 nmol thymidine hours(-1) g-1 dry weight), whereas activities decreased with depth (0.46 ± 0.21 nmol thymidine hours(-1) g(-I) dry weight) below sediment surface.

Phosphatidylcholine synthesis, secretion, and reutilization during differentiation of the surfactant-producing type II alveolar cell from fetal rabbit lungs.

Evidence indicates that pulmonary pool sizes of choline and related intermediates available for synthesis of phosphatidylcholine, the major component of the surfactant, change during gestation. Furthermore, recent data suggest that the type II lung cells that produce the surfactant potentially can reutilize components of this material. However, the relationship of the de novo synthetic mechanism to the secretion and reutilization of phosphatidylcholine has not been established. This is particularly true in the case of the fetal lung where, although alterations in precursor pool sizes, including choline, have been demonstrated, few or no data are available concerning how phosphatidylcholine synthesis affects or is affected by secretion and reutilization of this phospholipid by fetal type II cells. The present study was undertaken to determine the effect of availability of choline on de novo synthesis of phosphatidylcholine by isolated fetal rabbit type II cells during the differentiation process. In addition, differentiating type II alveolar cells were used to examine the hypothesis that these cells incorporate phospholipid from the extracellular environment and the quantity and/or composition of this phospholipid differently affects cellular secretion or de novo phosphatidylcholine synthesis. Assuming that the cells did not discriminate between radioactive and nonradioactive choline, elevation of extracellular choline increased the synthesis of cellular phosphatidylcholine and disaturated phosphatidylcholine in a dose-dependent manner to 0.08 mM choline. Cells induced to differentiated by exposure to fibroblast-conditioned medium synthesized more total and disaturated phosphatidylcholine at all extracellular choline concentrations. Incubation of the fetal type II cells with dipalmitoylphosphatidylcholine or 1-palmitoyl-2-oleoylphosphatidylcholine significantly depressed the incorporation of [3H]choline into cellular phosphatidylcholine after 24 or 48 h, but not necessarily at both times. Dipalmitoylphosphatidylcholine depressed the secretion of [3H]choline-labeled phosphatidylcholine after incubation for 24 h. 1-Palmitoyl-2-oleoylphosphatidylcholine stimulated the secretion of tritium-labeled phosphatidylcholine at a concentration of 25 micrograms/mL after 48 h. Comparison of the phospholipid effect by incubating the cells with 50 ng of 14C-labeled phospholipid for 24 h showed that 1-palmitoyl-2-oleoylphosphatidylcholine significantly reduced the synthesis of 3H-labeled phosphatidylcholine compared to dipalmitoylphosphatidylcholine. In contrast, 1-palmitoyl-2-oleoylphosphatidylcholine stimulated secretion of 3H-labeled phosphatidylcholine compared with the disaturated moiety. The differentiation state of the cells altered the magnitude of the cellular secretion response but not the character.(ABSTRACT TRUNCATED AT 400 WORDS)

Transforming Growth Factor-β Increases Elastin Production by Neonatal Rat Lung Fibroblasts

While elastin degradation is a hallmark of pulmonary emphysema, it is likely that elastin synthesis also occurs. However, the supramolecular structure and function of the newly synthesized elastin are abnormal. Very little is known about the regulation of elastin synthesis during the development of emphysema when prominent collections of mononuclear phagocytes are found in and near the alveolar interstitium. Transforming growth factor-beta (TGF-beta) is an important regulator of collagen and fibronectin production in wound healing, which is also accompanied by an influx of mononuclear phagocytes. We hypothesized that TGF-beta may influence elastin production by fibroblasts in the pulmonary interstitium. Therefore, we examined the influence of TGF-beta on the production of elastin by postconfluent cultures of neonatal rat lung fibroblasts. Elastin production was quantitated by analyzing the incorporation of [3H]valine into the soluble elastin precursor tropoelastin (TE). The incorporation of [3H]valine into TE was approximately 2-fold greater in the presence of 40 or 100 pM TGF-beta than in its absence. The intracellular, free [3H]valine pool was increased by 18% in the presence of TGF-beta. Therefore, TGF-beta-related differences in the precursor pool size were not solely responsible for the observed increase in [3H]valine incorporation. Northern analysis demonstrated that the increase in TE was accompanied by a smaller but significant increase in the steady-state level of elastin mRNA. Thus, the observed increase in TE production can be at least partially attributed to a pretranslational effect of TGF-beta.

Effects of chronic ethanol consumption on the synthesis of polypeptides encoded by the hepatic mitochondrial genome

Liver mitochondria from rats fed ethanol chronically demonstrate an impaired ability to incorporate [ 35S]methionine into polypeptide products in vitro. This ethanol-induced effect on mitochondrial translation in vitro could not be attributed to significant differences in the methionine precursor pool sizes of ethanol and control mitochondria or to the acute effects of residual ethanol. The observed reduction of radiolabeled methionine incorporation into mitochondrial gene products of ethanol mitochondria in vitro reflects a decrease in the synthesis of all the mitochondrial gene products. However, the percentage of total radiolabel incorporated into each gene product is unaffected by ethanol, suggesting an ethanol-induced coordinate depression of mitochondrial protein synthesis. Moreover, SDS-PAGE and densitometry of submitochondrial particles from ethanol-fed and control rats demonstrated that the steady-state concentration of each of the mitochondrial gene products is decreased in ethanol-fed rats. This reduction of the steady-state concentration of the mitochondrial gene products may be related to the observed depressions of oxidative phosphorylation activities associated with hepatic mitochondria from ethanol-fed rats.

Evidence for a Universal Pathway of Abscisic Acid Biosynthesis in Higher Plants from 18O Incorporation Patterns

Previous labeling studies of abscisic acid (ABA) with (18)O(2) have been mainly conducted with water-stressed leaves. In this study, (18)O incorporation into ABA of stressed leaves of various species was compared with (18)O labeling of ABA of turgid leaves and of fruit tissue in different stages of ripening. In stressed leaves of all six species investigated, avocado (Persea americana), barley (Hordeum vulgare), bean (Phaseolus vulgaris), cocklebur (Xanthium strumarium), spinach (Spinacia oleracea), and tobacco (Nicotiana tabacum), (18)O was most abundant in the carboxyl group, whereas incorporation of a second and third (18)O in the oxygen atoms on the ring of ABA was much less prominent after 24 h in (18)O(2). ABA from turgid bean leaves showed significant (18)O incorporation, again with highest (18)O enrichment in the carboxyl group. The (18)O-labeling pattern of ABA from unripe avocado mesocarp was similar to that of stressed leaves, but in ripe fruits there was, besides high (18)O enrichment in the carboxyl group, also much additional (18)O incorporation in the ring. In ripening apple fruit tissue (Malus domestica), singly labeled ABA was most abundant with more (18)O incorporated in the tertiary hydroxyl group than in the carboxyl group of ABA. Smaller quantities of this monolabeled product (C-1'-(18)OH) were also detected in the stressed leaves of barley, bean, and tobacco, and in avocado fruits. It is postulated that a large precursor molecule yields an aldehyde cleavage product that is, in some tissues, rapidly converted to ABA with retention of (18)O in the carboxyl group, whereas in ripening fruits and in the stressed leaves of some species the biosynthesis of ABA occurs at a slower rate, allowing this intermediate to exchange (18)O with water. On the basis of (18)O-labeling patterns observed in ABA from different tissues it is concluded that, despite variations in precursor pool sizes and intermediate turnover rates, there is a universal pathway of ABA biosynthesis in higher plants which involves cleavage of a larger precursor molecule, presumably an oxygenated carotenoid.

Induction of phosphatidylcholine biosynthesis via cdpcholine pathway in lung and liver of rats following intratracheal administration of ddt and endosulfan

The induction of phosphatidylcholine (PC) biosynthesis via the CDPcholine pathway in lung and liver of rats has been shown following the intratracheal administration of 1,1,1-trichloro-2m2-bis(p-chlorophenyl) ethane (DDT) (5 mg/100 g body weight) and endosulfan (1 mg/100 g body weight) for 3 days. Controls received only the vehicle solution (groundnut oil, 0.1 m1/100 g body weight). The treatment of DDT and endosulfan significantly increased the PC contents and the incorporation of radioactive [methyl-3H]choline into PC of lung and liver microsomes. The incorporation of radioactive [methyl-14C]methionine into microsomal PC of lung and liver was not affected significantly by treatment with either of the insecticides. 1,4,5,6,7-hexachloro-5-norbornene-2,3-dimethano cyclic sulfite (endosulfan) administration significantly increased the activity of choline kinase and phosphocholine cytidylyltransferase (both cytosolic and microsomal) of lung, whereas DDT increased the activity of only latter. In liver, both DDT and endosulfan administration significantly increased the activity of choline kinase and phosphocholine cytidylyltransferase (both cytosolic and microsomal). However, the activity of phosphocholinetransferase was not affected in both lung and liver microsomes of rats treated with these insecticides. The PC precursor pool sizes, choline and phosphorylcholine, of lung and liver tissues were not altered by DDT and endosulfan treatments. The present results suggest that the increased level of PC and incorporation of radioactive [methyl-3H]choline into microsomal PC could be the result of increased activity of choline kinase and phosphocholine cytidylyltransferase of lung and liver of rats following intratracheal administration of DDT and endosulfan.

Metabolic labelling of polyisoprenoids using tritiated water

Procedures are described for the metabolic labelling of polyisoprenoids using tritiated water. Unlike other precursors, tritiated water equilibrates rapidly with tissue water to generate a pool which remains constant for the duration of most experiments. The problems of differences in precursor pool size between tissues and physiological states are thus avoided. Results obtained for the metabolic labelling of dolichol and dolichyl phosphate (Dol-P) in soybean embryos and mice, both of which contain primarily dolichol, are presented. The methodology for soybeans was applied to rye which contains nearly equal amounts of dolichol and polyprenol. These alcohols (and presumably their phosphorylated forms) can also be metabolically labelled using tritiated water. The rye embryo provides an excellent system for studies on the α-saturation reaction responsible for the formation of dolichol intermediates from corresponding polyprenol forms.

Regulation of Phosphatidylcholine Biosynthesis in Activated Alveolar Type II Cells

The biosynthesis of phosphatidylcholine was studied in a population of activated Type II cells isolated from the lungs of rats treated with silica. Type II cells were separated by centrifugal elutriation into two populations, designated Type IIA and Type IIB. The Type IIB or activated population consisted of Type II cells that were larger than normal cells; Type IIA cells were morphologically similar to normal Type II cells. Type IIB cells incorporated more [Me-14C]choline into both total phosphatidylcholine and disaturated phosphatidylcholine than did Type IIA or control Type II cells. Measurement of the pool sizes of the choline-containing precursors to phosphatidylcholine indicated that the biosynthesis of phosphatidylcholine was increased 4- to 5-fold in Type IIB cells. Increased conversion of cholinephosphate to CDP-choline was associated with increased phosphatidylcholine biosynthesis in Type IIB cells. Cholinephosphate cytidylyltransferase activity was increased approximately threefold in Type IIB cells. Subcellular fractionation indicated that essentially all of the increase in cytidylyltransferase activity was associated with the particulate fraction (100,000 x g pellet). In Type IIB cells, the particulate fraction contained 83% of the total cellular cytidylyltransferase activity; in control cells, this fraction contained 67% of the total activity. The specific activity of the cytidylyltransferase associated with the particulate fraction was increased twofold in Type IIB cells. The specific activity of the cytosolic enzyme was similar to that in control cells. Cholinephosphotransferase specific activity was increased approximately twofold in the activated Type II cells. The specific activity of choline kinase was the same as that in control Type II cells. These results demonstrate that the increased biosynthesis of phosphatidylcholine in Type IIB cells is a result of stimulation of the CDP-choline pathway. This study indicates that both cholinephosphate cytidylyltransferase and cholinephosphotransferase may be involved in regulating the de novo biosynthesis of phosphatidylcholine in alveolar Type II cells.

Assembly kinetics and identification of precursor proteins of complex I from Neurospora crassa

Complex I from Neurospora crassa was fractionated using chaotropic agents and various chromatographic techniques. Several subunits were isolated. Polyclonal antibodies directed against the holocomplex or individual subunits were raised in rabbits, and employed to analyse the composition and assembly of this respiratory chain enzyme in vivo. N. crassa cells were pulse-labelled with radioactive amino acids. The time course of incorporation of radioactivity into complex-I polypeptides was studied by immunoprecipitation. The labelling kinetics of whole complex I was found to be similar to that of cytochrome oxidase, displaying a half-maximal labelling time of 10 min. Newly synthesized individual polypeptide subunits (about 23 species) assembled into the holoenzyme at markedly different rates. Two mitochondrially synthesized proteins, a 29-kDa polypeptide (the ND-1 gene product) and a 12-kDa polypeptide were the fastest components to appear in the enzyme. We estimate that the precursor pool sizes of all components range between 1-25% of the amounts present in the final complex. Precursors of polypeptides of complex I were synthesized in an heterologous cell-free system and immunoprecipitated with subunit specific antibodies. Six isolated precursors were compared with the corresponding mature proteins. It appears that four subunits (apparent molecular masses of 22, 25, 31 and 33 kDa) are initially synthesized as larger-molecular-mass precursors. Two subunits (apparent molecular masses of 12.5 and 14 kDa) are made with the same size as their mature forms.

High-performance liquid-chromatographic analysis of bases and nucleoside in mouse testes following in vivo thymidine administration

In order to measure accurately bases and nucleosides in vivo, a method has been developed using high-performance liquid chromatography. This method is reproducible under conditions of constant pressure, temperature and pH and is rapid, requiring approximately 1 h for a complete chromatographic separation of bases and nucleosides. The method has been used to measure quantitatively the bases and nucleosides in mouse testes following in vivo administration of thymidine. Groups of male mice were given thymidine by intra-peritoneal injection and were killed at various time intervals and the testes removed for extraction procedures. There was a significant increase in the thymidine concentration in the testes 1 h after injection, which fairly quickly returned to the control value thereafter. There was a subsequent reduction in uridine concentration. This analytical method allows deoxyribonucleotide (dNTP) precursor pool sizes to be determined in germinal cells. It may also be useful for other tissues in vivo.

Early metabolic responses of retinal neurons to trimethyltin intoxication.

Chronic systemic exposure of rats to the neuronotoxic compound trimethyltin (TMT) results in increased incorporation of radioactive precursors into retinal proteins and glycoproteins. Because this increased metabolic activity is accompanied by minimal subcellular pathological alterations and almost no neuronal necrosis, we suggested that it may represent an early, reactive (compensatory) response (Brain Res. 398, 298-304; 1986). We have now investigated the development of this metabolic response to TMT in more detail. Beginning at 30 d of age, rats received weekly doses of TMT (4 mg/kg body wt) by gavage for up to 7 wk; rates of incorporation of [35S]methionine and [3H]fucose into retinal proteins and glycoproteins, respectively, were then determined using in vitro retinal incubations. The apparent rates of protein synthesis and glycoprotein glycosylation in retinas from TMT-treated animals were normal or slightly decreased after 1-3 wkly doses, but were increased after 4 doses and more markedly increased after 7 doses. Glycoprotein glycosylation was increased to a greater degree (192% of control after 7 wk of dosing) than was protein synthesis (134% of control). The increased incorporation in retinas from TMT-treated animals persisted when retinas were incubated with "flooding" concentrations of precursor (1 mM), suggesting that these increases were not owing to alterations in the size of retinal precursor pools. The preferential increase in glycoprotein glycosylation was partially owing to a selective increase in glycosylation of two molecular species with apparent mol wt of 32 and 45 KDa. Quantitative autoradiographic analysis of newly synthesized proteins and glycoproteins indicated that the TMT-induced increase in metabolic activity was not specific or selective for any retinal layer or cell type. We suggest that the preferential activation of glycoprotein glycosylation, and in particular the increased glycosylation of the 32 and 45 KDa glycoprotein species, may represent part of a compensatory metabolic response of retinal neurons to TMT-induced neuronal injury.

Effects of dietary conditions on the pool sizes of precursors of phosphatidylcholine and phosphatidylethanolamine synthesis in rat liver

We developed a new method for the determination of choline- and ethanolamine-containing precursors of phosphatidylcholine and phosphatidylethanolamine after their separation by HPLC and we have studied the effects of different dietary conditions on the pool sizes of these metabolites in rat liver. Fasting for 48 h induced only a small decrease in the amounts of phosphatidylethanolamine and its water-soluble precursors. Upon refeeding with a high-sucrose, fat-free diet for 72 h, the levels of ethanolamine-containing compounds were only slowly restored. The effects of various dietary conditions on the amounts of phosphatidylcholine and its water-soluble precursors were much more pronounced. Fasting induced a sharp decrease, especially of the amount of cholinephosphate. However, the levels of phosphatidylcholine and the choline-containing precursors were rapidly restored upon refeeding for 24 h. Continued refeeding for an additional 48 h enhanced the cholinephosphate pool size to a level more than double that found in normally fed rats. The latter effect was accompanied by an inhibition of the enzyme CTP: choline-phosphate cytidylyltransf erase. The results are discussed in view of a possible regulatory mechanism that may balance the amounts of phosphatidylcholine and phosphatidylethanolamine.

Variation in precursor pool size during the division cycle of Escherichia coli: further evidence for linear cell growth

The magnitudes of several pools of radioactively labeled precursors for RNA and protein synthesis were determined as a function of cell age during the division cycle of Escherichia coli 15 THU. Uracil, histidine, and methionine pools increased from low initial values for cells at birth to maxima during midcycle and then subsided again. These pools were small or nonexistent at the beginning and the end of the cycle, and their average values during the cycle were less than 4% of the total cellular radioactivity. The results are consistent with a linear pattern of growth for cells during the division cycle and provide strong evidence against exponential or bilinear growth of E. coli cells.

Tunicamycin inhibits proteoglycan synthesis in rat ovarian granulosa cells in culture

The effects of tunicamycin, an inhibitor of N-linked oligosaccharide biosynthesis, on the synthesis and turnover of proteoglycans were investigated in rat ovarian granulosa cell cultures. The synthesis of proteoglycans was inhibited (40% of the control at 1.6 μg/ml tunicamycin) disproportionately to that of general protein synthesis measured by [ 3H]serine incorporation (80% of control). Proteoglycans synthesized in the presence of tunicamycin lacked N-linked oligosaccharides but contained apparently normal O-linked oligosaccharides. The dermatan sulfate and heparan sulfate chains of the proteoglycans had the same hydrodynamic size as control when analyzed by Sepharose 6B chromatography. However, the disulfated disaccharide content of the dermatan sulfate chains was reduced by tunicamycin in a dose-dependent manner, implying that the N-linked oligosaccharides may be involved in the function of a sulfotransferase which is responsible for sulfation of the iduronic acid residues. When [ 35S]sulfate and [ 3H]glucosamine were used as labeling precursors, the ratio of 35S 3H in chondroitin 4-sulfate was reduced to approximately 50% of the control by tunicamycin, indicating that the drug reduced the supply of endogenous sugar to the UDP- N-acetylhexosamine pool. Neither transport of proteoglycans from Golgi to the cell surface nor their turnover from the cell surface (release into the medium, or internalization and subsequent intracellular degradation) was affected by the drug. Addition of mannose 6-phosphate to the culture medium did not alter the proteoglycan turnover. When granulosa cells were treated with cycloheximide, completion of proteoglycan diminished with a t 1 2 of approximately 12 min, indicating the time required for depleting the core protein precursor pool. The glycosaminoglycan synthesizing capacity measured by the addition of p-nitrophenyl-β-xyloside, however, lasted longer ( t 1 2 of approximately 40 min). Tunicamycin decreased the core protein precursor pool size in parallel to decreased proteoglycan synthesis, both of which were significantly greater than the inhibition of general protein synthesis. This suggests two possibilities: (a) tunicamycin specifically inhibited the synthesis of proteoglycan core protein, or more likely (b) a proportion of the synthesized core protein precursor (approximately 50%) did not become accessible for post-translational modifications, and was possibly routed for premature degradation.

Effects of acipimox, a nicotinic acid derivative, on lipolysis in human adipose tissue and on cholesterol synthesis in human jejunal mucosa.

The mode of action of acipimox (5-methyl-pyrazine carboxylic acid 4-oxide), an hypotriglyceridaemic agent, was examined in human adipose tissue and intestinal mucosa. The rates of release of fatty acids and glycerol from human adipose tissue were measured in vitro. The release of fatty acids and glycerol from adipose tissue maximally stimulated by isoprenaline (10(-5) mol/l) fell by 40 and 25% respectively (P less than 0.025 and P less than 0.025) in the presence of acipimox (10(-5) mol/l). In submaximally stimulated adipose tissue (isoprenaline 10(-7) mol/l) acipimox (10(-4) mol/l) fully inhibited release of fatty acids (P less than 0.05) and glycerol (P less than 0.025) to basal rates. In unstimulated adipose tissue acipimox (10(-3) mol/l) reduced the rate of glycerol release (P less than 0.05), but not the rate of fatty acid release. Cholesterol synthesis in jejunal mucosa was measured in vitro by the incorporation of [2-14C]-acetate into sterols. Addition of cholesterol to the incubation reduced [2-14C]acetate incorporation into sterols from 8.7 +/- 2.1 (mean +/- standard error) to 3.7 +/- 1.0 pmol h-1 mg-1 of tissue (P less than 0.01). Acipimox at 10(-4)-10(-2) mmol/l had no consistent effect on cholesterol synthesis. Acipimox appears to exert its main hypolipidaemic effect by reducing lipolysis and free fatty acid flux to the liver, thereby reducing the precursor pool size of very low density lipoprotein (VLDL)-triglyceride and VLDL synthesis.

Pool sizes of precursors for phosphatidylcholine formation in adult rat lung type II cells

1. 1. The pool sizes of the choline intermediates and cofactors involved in the CDPcholine pathway were studied in alveolar type II cells from adult rat lung. 2. 2. The choline phosphate pool was much larger than both the choline and CDPcholine pools. 3. 3. Kinetic analysis of the pool sizes revealed that cholinephosphate cytidylyltransferase catalyzes a non-equilibrium reaction. 4. 4. These results are consistent with the idea that cholinephosphate cytidylyltransferase regulates the choline flux into phosphatidylcholine in adult rat lung type II cells.

Effect of trichothecenes on growth and intracellular pool size of Mycoplasma gallisepticum

The mycotoxin T-2 inhibited the growth of Mycoplasma gallisepticum. The growth inhibition was most pronounced with the hydrophobic derivatives T-2 acetate and very little with the hydrophilic T-2 tetraol. The toxin had no effect on the biosynthesis of either protein, DNA, RNA or complex lipids but markedly reduced the intracellular pool size of soluble low molecular mass precursors. It seems that T-2 acetate, by virtue of its hydrophobic nature, may accumulate within the lipid backbone affecting the permeability properties of the cell membrane. T-2 toxin Mycoplasma gallisepticum Mycoplasma membrane Trichothecen Mycotoxin Intracytoplasmic pool