Rate Of Net Synthesis Research Articles

Renovating a double fence with or without notifying the next door and across the street neighbors: why the biogenic cytoplasmic membrane of Gram-negative bacteria display asymmetry?

The complex two-membrane organization of the envelope of Gram-negative bacteria imposes an unique biosynthetic and topological constraints that can affect translocation of lipids and proteins synthesized on the cytoplasm facing leaflet of the cytoplasmic (inner) membrane (IM), across the IM and between the IM and outer membrane (OM). Balanced growth of two membranes and continuous loss of phospholipids in the periplasmic leaflet of the IM as metabolic precursors for envelope components and for translocation to the OM requires a constant supply of phospholipids in the IM cytosolic leaflet. At present we have no explanation as to why the biogenic E. coli IM displays asymmetry. Lipid asymmetry is largely related to highly entropically disfavored, unequal headgroup and acyl group asymmetries which are usually actively maintained by active mechanisms. However, these mechanisms are largely unknown for bacteria. Alternatively, lipid asymmetry in biogenic IM could be metabolically controlled in order to maintain uniform bilayer growth and asymmetric transmembrane arrangement by balancing temporally the net rates of synthesis and flip-flop, inter IM and OM bidirectional flows and bilayer chemical and physical properties as spontaneous response. Does such flippase-less or 'lipid only", 'passive' mechanism of generation and maintenance of lipid asymmetry exists in the IM? The driving force for IM asymmetry can arise from the packing requirements imposed upon the bilayer system during cell division through disproportional distribution of two negatively curved phospholipids, phosphatidylethanolamine and cardiolipin, with consistent reciprocal tendency to increase and decrease lipid order in each membrane leaflet respectively.

PKPD modelling to predict altered disposition of 1α,25-dihydroxyvitamin D3 in mice due to dose-dependent regulation of CYP27B1 on synthesis and CYP24A1 on degradation.

Concentrations of 1α,25-dihydroxyvitamin D3 [1,25(OH)2 D3 ], the active ligand of the vitamin D receptor, are tightly regulated by CYP27B1 for synthesis and CYP24A1 for degradation. However, the dose-dependent pharmacokinetic (PK)-pharmacodynamic (PD) relationship between these enzymes and 1,25(OH)2 D3 concentrations has not been characterized. The pharmacokinetics of 1,25(OH)2 D3 were evaluated after administration of single (2, 60 and 120 pmol) and repeated (2 and 120 pmol q2d ×3) i.v. doses to male C57BL/6 mice. mRNA expression of CYP27B1 and CYP24A1 was examined by quantitative PCR and 1,25(OH)2 D3 concentrations were determined by enzyme immunoassay. CYP27B1 and CYP24A1 changes were absent for the 2 pmol dose and biexponential decay profiles showed progressively shorter terminal half-lives with increasing doses. Fitting with a two-compartment model revealed decreasing net synthesis rates and increasing total clearances with dose, consistent with a dose-dependent down-regulation of renal CYP27B1 and the induction of renal/intestinal CYP24A1 mRNA expression. Upon incorporation of PD parameters for inhibition of CYP27B1 and induction of CYP24A1 to the simple two-compartment model, fitting was significantly improved. Moreover, fitted estimates for the 2 pmol dose, together with the PD parameters as modifiers, were able to predict profiles reasonably well for the higher (60 and 120 pmol) doses. Lastly, an indirect response model, which considered the synthesis and degradation of enzymes, adequately described the PK and PD profiles. The unique PK of exogenously administered 1,25(OH)2 D3 led to changes in PD of CYP27B1 and CYP24A1, which hastened the clearance of 1,25(OH)2 D3 .

Triglycerides produced in the livers of fasting rabbits are predominantly stored as opposed to secreted into the plasma

ObjectiveThe liver plays a central role in regulating fat metabolism; however, it is not clear how the liver distributes the synthesized triglycerides (TGs) to storage and to the plasma. Materials and methodsWe have measured the relative distribution of TGs produced in the liver to storage and the plasma by means of U-13C16-palmitate infusion in anesthetized rabbits after an overnight fast. ResultsThe fractional synthesis rates of TGs stored in the liver and secreted into the plasma were not significantly different (stored vs. secreted: 31.9±0.8 vs. 27.7±2.6%∙h−1, p>0.05). However, the absolute synthesis rates of hepatic stored and secreted TGs were 543±158 and 27±7nmol∙kg−1∙min−1 respectively, indicating that in fasting rabbits the TGs produced in the liver were predominately stored (92±3%) rather than secreted (8±3%) into the plasma. This large difference was mainly due to the larger pool size of the hepatic TGs which was 21±9-fold that of plasma TGs. Plasma free fatty acids (FFAs) contributed 47±1% of the FA precursor for hepatic TG synthesis, and the remaining 53±1% was derived from hepatic lipid breakdown and possibly plasma TGs depending on the activity of hepatic lipase. Plasma palmitate concentration significantly correlated with hepatic palmitoyl-CoA and TG synthesis. ConclusionIn rabbits, after an overnight fast, the absolute synthesis rate of hepatic stored TGs was significantly higher than that of secreted due to the larger pool size of hepatic TGs. The net synthesis rate of TG was approximately half the absolute rate. Plasma FFA is a major determinant of hepatic TG synthesis, and therefore hepatic TG storage.

Butyrate ingestion improves hepatic glycogen storage in the re-fed rat

BackgroundButyrate naturally produced by intestinal fiber fermentation is the main nutrient for colonocytes, but the metabolic effect of the fraction reaching the liver is not totally known. After glycogen hepatic depletion in the 48-hour fasting rat, we monitored the effect of (butyrate 1.90 mg + glucose 14.0 mg)/g body weight versus isocaloric (glucose 18.2 mg/g) or isoglucidic (glucose 14.0 mg/g) control force-feeding on in vivo changes in hepatic glycogen and ATP contents evaluated ex vivo by NMR in the isolated and perfused liver.ResultsThe change in glycogen was biphasic with (i) an initial linear period where presence of butyrate in the diet increased (P = 0.05) the net synthesis rate (0.20 ± 0.01 μmol/min.g-1 liver wet weight, n = 15) versus glucose 14.0 mg/g only (0.16 ± 0.01 μmol/min.g-1 liver ww, n = 14), and (ii) a plateau of glycogen store followed by a depletion. Butyrate delayed the establishment of the equilibrium between glycogenosynthetic and glycogenolytic fluxes from the 6th to 8th hour post-feeding. The maximal glycogen content was then 97.27 ± 10.59 μmol/g liver ww (n = 7) at the 8th hour, which was significantly higher than with the isocaloric control diet (64.34 ± 8.49 μmol/g, n = 12, P = 0.03) and the isoglucidic control one (49.11 ± 6.35 μmol/g liver ww, n = 6, P = 0.003). After butyrate ingestion, ATP content increased from 0.95 ± 0.29 to a plateau of 2.14 ± 0.23 μmol/g liver ww at the 8th hour post-feeding (n = 8) [P = 0.04 versus isoglucidic control diet (1.45 ± 0.19 μmol/g, n = 8) but was not different from the isocaloric control diet (1.70 ± 0.18 μmol/g, n = 12)].ConclusionThe main hepatic effect of butyrate is a sparing effect on glycogen storage explained (i) by competition between butyrate and glucose oxidation, glucose being preferentially directed to glycogenosynthesis during the post-prandial state; and (ii) by a likely reduced glycogenolysis from the newly synthesized glycogen. This first demonstration of the improvement of liver glycogen storage by acute butyrate supply may be an important contribution to explaining the beneficial effects on glucose homeostasis of nutritional supply increasing butyrate amount such as fiber diets.

IFN-gamma gene therapy by intrasplenic hepatocyte transplantation: a novel strategy for reversing hepatic fibrosis in Schistosoma japonicum-infected mice.

Liver-targeted gene therapy using hepatocyte as recipient cells has recently been documented to be effective in treatment of numerous hepatic diseases, such as metabolic diseases and liver carcinoma. IFN-gamma elicits antipreliferative and antifibrogenic activity in a variety of mesenchymal cells, including hepatic satellite cells. To investigate the antifibrogenic response of liver gene therapy mediated by intrasplenic transplantation of gene-modified hepatocytes, normal mouse liver cell line BNL CL.2 cells were transfected with murine IFN-gamma gene (BNL.IFN-gamma) in vitro, and transplanted intrasplenically into Schistosoma japonicum-infected mice. The amounts and distribution of IFN-gamma (which inhibits collagen synthesis), TGF-beta (which stimulates collagen synthesis) and extracellular matrix, including type I and III collagen, were detected. In mice infected with S. japonicum and then treated with BNL.IFN-gamma, an increase of IFN-gamma and decrease of TGF-beta1 were detected at 20 weeks postinfection compared to untreated S. japonicum-infected mice. Immunohistochemical analysis showed that S. japonicum infection induced a marked increase of type I and III collagen synthesis. Whereas, 4 weeks after treatment with BNL.IFN-gamma, net synthesis rates of type I and III collagen were markedly decreased in the liver of infected mice. In addition, a decreased expression of TGF-beta1 and its receptor TGF-betaRII in the liver of BNL.IFN-gamma-treated mice was also observed. Moreover, the decrease in TGF-beta1 and TGF-betaRII protein approximately paralleled the decrease in their mRNA expression, which was detected by RNA dot blotting. The data indicate that intrasplenic transplantation of IFN-gamma gene-modified hepatocyte can be a candidate approach to treat hepatic fibrosis.

GENETIC AND PHYSIOLOGICAL VARIATION IN PIGMENT COMPOSITION OF EMILIANIA HUXLEYI (PRYMNESIOPHYCEAE) AND THE POTENTIAL USE OF ITS PIGMENT RATIOS AS A QUANTITATIVE PHYSIOLOGICAL MARKER.

Genetic variation of pigment composition was studied in 16 different strains of Emiliania huxleyi (Lohm.) Hay et Mohler in batch culture. Distinct strain-dependent differences were found in the ratios of fucoxanthin, 19'-hexanoyloxyfucoxanthin, and 19'-butanoyloxyfucoxanthin, hampering the use of these individual pigments as a taxonomic marker at the species level. The molar ratio of total carotenoids to chl a, however, was constant for all strains tested. In addition, the pigment composition of one axenic strain (L) of E. huxleyi at different growth rates in light-, nitrate-, and phosphate-limited continuous cultures was analyzed quantitatively. The pigments fucoxanthin and 19'-hexanoyloxyfucoxanthin correlated closely under all conditions. From steady-state rate calculations, it is hypothesized that 19'-hexanoyloxyfucoxanthin is synthesized from fucoxanthin, with light as a modulating factor. The net rate of synthesis of diatoxanthin depended both on the concentration of diadinoxanthin (its partner in the xanthophyll cycle) and on light, illustrating its photoprotective function in the xanthophyll cycle. In axenic strain L, the ratio of total fucoxanthins to chl a correlated strongly with photon flux density and can potentially be used to assess the physiological status with respect to irradiance in field populations. In multispecific bloom situations, the ratio of diadinoxanthin plus diatoxanthin to total fucoxanthins could be used as an alternative indicator for the light-dependent physiological state of E. huxleyi, provided that no other chromophytes are present. Application of these correlations to mesocosm data from the literature has so far provided no evidence that E. huxleyi blooms form only at inhibiting light levels, as previously suggested.

Manipulation of cholesterol and cholesteryl ester synthesis has multiple effects on the metabolism of apolipoprotein B and the secretion of very-low-density lipoprotein by primary hepatocyte cultures

Inhibition of esterified and non-esterified cholesterol synthesis by lovastatin in primary rat hepatocytes suppressed the net synthesis and very-low-density lipoprotein (VLDL) secretion of apolipoprotein B (apoB)-48 and apoB-100. Lovastatin did not alter the rates of apoB-48 and apoB-100 post-translational degradation. 25-Hydroxycholesterol, which inhibited non-esterified cholesterol synthesis but increased the synthesis of cholesteryl ester, showed differential effects on the metabolism of apoB-48 and apoB-100. Whereas the secretion of apoB-48 VLDL was suppressed there was no effect on the secretion of apoB-100 VLDL. The post-translational degradation of apoB-48, but not of apoB-100, was enhanced by 25-hydroxycholesterol. The net synthesis rates of apoB-48 and apoB-100 were unaffected by 25-hydroxycholesterol. The inhibitory effect of lovastatin alone on the net synthesis of apoB-48 and apoB-100 was reversed by the simultaneous presence of 25-hydroxycholesterol, suggesting a role for newly synthesised cholesteryl ester. Prevention of the reversal effect by the acyl-CoA: cholesterol acyltransferase (ACAT) inhibitor YM 17E supported this interpretation. In the presence of lovastatin, restoration of the net synthesis of apoB by 25-hydroxycholesterol was not accompanied by an increased VLDL output of apoB-48 and apoB-100. However, under these conditions there was an increased post-translational degradation of apoB-48 and apoB-100. These results suggest that interference with intracellular cholesterol and cholesteryl ester metabolism interrupts VLDL assembly at sites of both apoB net synthesis and post-translational degradation.

Dietary carbohydrate and postexercise synthesis of proglycogen and macroglycogen in human skeletal muscle.

This study examined the role of carbohydrate (CHO) ingestion on the resynthesis of two pools of glycogen, proglycogen (PG) and macroglycogen (MG), in human skeletal muscle. Nine males completed an exhaustive glycogen depletion exercise bout at 70% maximal O2 consumption on two occasions. Subsequent 48-h dietary interventions consisted of either high (HC, 75% of energy intake) or low (LC, 32% of energy intake) CHO diets. Muscle biopsies were taken at exhaustion (EXH) and 4, 24, and 48 h later. The total muscle glycogen (Gt) at EXH for the HC and LC conditions was not significantly different, and the MG represented approximately 12% of the Gt. From EXH to 4 h, there was an increase in the PG only for HC and no change in MG in either diet (P < 0.05). From 4 to 24 h, the concentration of PG increased in both conditions (P < 0.05). Between 24 and 48 h, in HC the majority of the increase in Gt was due to the MG pool (P < 0.05). The MG and PG concentrations for HC were significantly greater than for LC at 24 and 48 h (P < 0.05). At 48 h the MG represented 40% of the Gt for the HC diet and only 21% for the LC diet. There was no change in the net rates of synthesis of PG or MG over 48 h for LC (P < 0.05). The net rate of PG synthesis from 0 to 4 h for HC was 16 +/- 1.68 mmol glucosyl units . kg dry wt-1 . h-1, which was threefold greater than for LC (P < 0. 05). The net rate of PG synthesis decreased significantly from 4 to 24 h for HC, whereas the net rate of MG synthesis was not different over 48 h but was significantly greater than in LC (P < 0.05). The two pools are synthesized at very different rates; both are sensitive to CHO, and the supercompensation associated with HC is due to a greater synthesis in the MG pool.

EFFECT OF HIGH AND LOW CARBOHYDRATE DIET ON THE SYNTHESIS OF PROGLYCOGEN AND MACROGLYCOGEN IN HUMAN SKELETAL MUSCLE

1471 The present study examined the role of carbohydrate(CHO) on the resynthesis of the two pools of glycogen, proglycogen(PG) and macroglycogen(MG), in human skeletal muscle. Ten subjects completed an exhaustive glycogen depletion exercise bout at 70% VO2 max on two occasions separated by 2 weeks. The diet was controlled by providing weighed, pre-packaged food such that either 75%(HC) or 32%(LC) of the energy was CHO. Muscle biopsies were taken at exhaustion(EXH), 4, 24 and 48 hours. The total muscle glycogen(TotG) at EXH was 79±15.6 and 113±20.1mmol glucosyl units/kg dw for HC and LC respectively. At exhaustion the MG concentration represented about 12% in each group. From EXH-4h there was an increase in the PG only for HC, and no change in MG in either diet(p<0.05). From 4-24h the concentration of PG increased in both conditions(p<0.05). Between 24-48h in HC all of the increase in TotG was due to the MG fraction(p<0.05). The MG and PG concentrations for HC were significantly greater than in LC at 24 and 48h (p<0.05). At 48h the MG represented 40% of the TotG for the HC diet and only 21% for LC. There was no change in the net rates of synthesis of PG or MG over 48h in LC(p<0.05). The net rate of PG synthesis from 0-4h in the HC was 16±1.68mmol glucosyl units/kg dw/h which is 3-fold greater than in the LC(p<0.05). The rate of PG synthesis decreased significantly 4-24h while the rate of MG synthesis was not significantly different over 48h but was significantly greater than LC(p<0.05). It appears that the two pools are dynamic and sensitive to CHO, especially the PG pool. The glycogen supercompensation is first generated in PG and the final increase in TotG occurs exclusively in MG. These data suggest that the MG pool is an important reservoir for glycogen synthesis at later time points if the CHO availability is high.

Stability of cyclin B protein during meiotic maturation and the first mitotic cell division in mouse oocytes

This report examines in detail the metabolism of the cyclin protein B1 during meiotic maturation and following the activation of mature mouse oocytes using immunoprecipitation of the radiolabelled protein. The net synthesis of cyclin B increases progressively during meiotic maturation, reaching its maximum levels at least 1 h before oocytes exit into metaphase of meiosis II (MII). This increase correlates with the rise in cdc2 kinase activity reported previously and suggests an association between the length of the first meiotic M phase (MI) and the net synthesis of cyclin B, that seems to regulate the time required for the cdc2 kinase to reach its maximum activity. Moreover, no marked degradation of cyclin B was observed before the MI to MII transition and that which occurs does so independently of the presence of microtubules, which are essential for cyclin degradation during metaphase II arrest and exit of oocytes into interphase of the first mitotic cell cycle. Cyclin B is degraded rapidly during the transitions MI to MII, MII to the first mitotic interphase and MII to an abortive third metaphase state (MIII). However, whilst its degradation was incomplete during the MI to MII transition, virtually no cyclin B protein was detected following both the MII to interphase and MII to MIII transitions. Thus, the decision of oocytes to exit into MIII, or interphase is not controlled at the level of cyclin B degradation. Lastly, in aging, non-activated oocytes, the net synthesis of cyclin B declines. Whereas, in activated eggs cultured in parallel although the rate of net synthesis declines initially, it is effectively 'rescued' being two-fold greater than in non-activated oocytes of an equivalent age. This gradual fall in the net synthesis of cyclin B observed in aging oocytes may contribute to the increasing ease with which they become activated, compared to recently ovulated oocytes.

Stability of cyclin B protein during meiotic maturation and the first mitotic cell division in mouse oocytes

This report examines in detail the metabolism of the cyclin protein B1 during meiotic maturation and following the activation of mature mouse oocytes using immunoprecipitation of the radiolabelled protein. The net synthesis of cyclin B increases progressively during meiotic maturation, reaching its maximum levels at least 1 h before oocytes exit into metaphase of meiosis II (MII). This increase correlates with the rise in cdc2 kinase activity reported previously and suggests an association between the length of the first meiotic M phase (MI) and the net synthesis of cyclin B, that seems to regulate the time required for the cdc2 kinase to reach its maximum activity. Moreover, no marked degradation of cyclin B was observed before the MI to MII transition and that which occurs does so independently of the presence of microtubules, which are essential for cyclin degradation during metaphase II arrest and exit of oocytes into interphase of the first mitotic cell cycle. Cyclin B is degraded rapidly during the transitions MI to MII, MII to the first mitotic interphase and MII to an abortive third metaphase state (MIII). However, whilst its degradation was incomplete during the MI to MII transition, virtually no cyclin B protein was detected following both the MII to interphase and MII to MIII transitions. Thus, the decision of oocytes to exit into MIII, or interphase is not controlled at the level of cyclin B degradation. Lastly, in aging, non-activated oocytes, the net synthesis of cyclin B declines. Whereas, in activated eggs cultured in parallel although the rate of net synthesis declines initially, it is effectively ‘rescued’ being two-fold greater than in non-activated oocytes of an equivalent age. This gradual fall in the net synthesis of cyclin B observed in aging oocytes may contribute to the increasing ease with which they become activated, compared to recently ovulated oocytes.

Transport of metal micronutrients in the phloem of castor bean (Ricinus communis) seedlings

The metal micronutrients (MN) copper, iron, manganese, and zinc are transported via the phloem in the course of remobilization and circulation. The extent of these processes and transport species are still largely unknown. The Ricinus seedling was used to study the transport of these metal micronutrients as well as their interactions with the plant‐endogenous chelator nicotianamine (NA) by daily measurements of the concentrations in the seedling parts and in the sieve tube sap obtained from a cut at the hypocotyl hook.The concentrations of these micronutrients in the phloem exudate decreased slightly from day 4 to day 8 of seedling development. Maximum values at day 4 were 65 μM for Zn, 63 μM for Fe, 27 μM for Cu, and 12 μM for Mn. The phloem transport rates reached maxima of 0.12 nmol cm−2h−1 for Zn and Fe at days 6 and 7, corresponding to the maximum exudation rates. The magnitude of these transport rates were in agreement with the net translocation rates estimated by analyses of the concentrations in the individual seedling parts.The NA content of the seedlings increased from day 0 (seed before sowing) until day 8, from 16 nmol to 474 nmol, which corresponds to an average net synthesis rate of about 100 nmol day−1 between the days 4 and 8. The NA:MN ratio was constant at 0.5 in the seedlings within this period. The NA concentrations and the sum of the concentrations of all four micronutrients in the sieve tube sap showed a constant ratio of 1.25 over the entire experimental period. Thus, both complex partners were subject to a cotransport in the phloem.Removal of the supplying endosperm led to a decrease in MN and NA concentrations in the sieve tube sap to about 80% while an average excess of NA of 1.1 was maintained. Since the concentrations of other amino acids, also possible chelators of metal micronutrients, fall to about 10% after removal of the endosperm, their role seems to be negligible as vehicles of MN transport in the phloem. Thus it is suggested that the divalent micronutrients considered in this study are loaded and maybe transported as NA complexes.

VISCOUS POLYSACCHARIDE AND STARCH SYNTHESIS IN RHODELLA RETICULATA (PORPHYRIDIALES, RHODOPHYTA)1

ABSTRACT Rhodella reticulata Deason, Butler and Rhyne produces copious amounts of a viscous polysaccharide (VP) during growth in batch cultures. The VPs accumulated on the cell surface and in the culture medium once cells ceased growth; starch concurrently accumulated within the cells. Light‐saturated 14C‐uptake declined steadily as the cells aged. Net synthesis rates for starch and mucilage were two‐ and four‐fold lower, respectively, in non‐growing cells than in growing cells, while the relative partitioning of newly‐fixed carbon into these materials was not different. These data suggest that total photosynthetic loading, rather than partitioning into one specific pool, controls cellular synthesis rates. No preferential synthesis of VPs occurred during the stationary phase. The findings have important implications for the commercial production of VPs.

Regulation of glycerol and starch metabolism in Chlamydomonas pulsatilla in response to changes in salinity

Abstract. Chlamydomonas pulsatilla rapidly adjusts its level of osmoregulatory solute, glycerol, when exposed to increased (upshock) or decreased (downshock) salinities. Rates of degradation, or loss, of glycerol are similar in light and dark, while rates of net synthesis proceed about 20% faster in the light than in the dark. Synthesis of glycerol in the dark is correlated with degradation of starch, and starch appears also to be utilized for glycerol synthesis in response to extensive salinity upshocks in the light, where photosynthesis is strongly inhibited. Under conditions of moderate upshocks in the light, photosynthesis is not affected, and net increases in both glycerol and starch were observed. Glycerol turnover takes place both under iso‐osmotic conditions, and during period of adjustments to osmotic downshocks, where there is a rapid net decrease in the glycerol pool. Half times for glycerol turnover were estimated from rates of incorporation of photoassimilated 14C into glycerol and glycerol pool sizes to be 20 and 74 min for cells incubated at 50 and 100% artificial seawater, respectively.

Effect of glucose starvation on glucose transport in neuronal cells in primary culture from rat brain.

The regulation of glucose transport into cultured brain cells during glucose starvation was studied. On glucose deprivation for 40 h, 2-deoxy-D-glucose (2-DG) uptake was stimulated twofold in neuronal cells but was not changed significantly in astrocytes. On refeeding, the increased activity of neuronal cells rapidly returned to the basal level, an observation indicating that the effect of glucose starvation was reversible. The increase was due solely to change in the Vmax, a finding suggesting that the number of glucose transporters on the plasma membrane is increased in starved cells. Cycloheximide inhibited this increase. In the presence of cycloheximide, the activity of 2-DG uptake of starved cells remained constant for 12 h and then slowly decreased, whereas that of fed cells decreased rapidly. These findings suggest that glucose starvation regulates glucose transport by changing the rate of net synthesis of the transporter in neuronal cells in culture.

Effect of dietary protein level on nitrogen metabolism in the growing bovine: I. Nitrogen recycling and intestinal protein supply in calves.

Eight Angus heifer calves (234 kg) were assigned to either a high (HP; 126 g N/d) or low (LP; 66.5 g N/d) protein intake. Calves received 4.8 kg DM/d consisting of 30% cottonseed hulls and 70% corn-soybean meal in equal portions at 4-h intervals. Single doses of 14C- and 15N-urea and 15N-ammonium sulfate were injected into the blood urea-N (BUN) and ruminal NH3-N (RAN) pools, respectively, to measure rate of flux through, and transfer of N between, these and bacterial N. Nitrogen balance was greater (P less than .05) for HP than for LP (56.9 vs 25.1 g N/d), but abomasal N flow as a percentage of N intake was greater (P less than .05) for LP than for HP (124 vs 71.1%). Pool size and net synthesis rate for both RAN and BUN pools were greater (P less than .05) for HP than for LP. Calves fed HP degraded more (P less than .05) BUN in the gastrointestinal tract than calves fed LP (37.4 vs 14.0 g N/d). Quantities of RAN absorbed from the rumen also were greater (P less than .05) for HP than for LP (14.2 vs 2.8 g N/d). The proportion of total gastrointestinal BUN degradation occurring in the rumen averaged 53 and 26% for LP and HP. Data are interpreted to suggest that net incorporation of BUN into bacterial protein (urea recycling) is inversely related to level of protein intake.

Substance P content in cultured neonatal rat vagal sensory neurons: the effect of nerve growth factor

To begin to study the factors regulating the synthesis and release of substance P (SP) in the sensory vagus nerve, cultures of neonatal rat nodose ganglia were developed. In microexplant cultures, obtained from small fragments of nodose ganglia, SP was present in low amounts: after 3 weeks,141 ± 36pg per well, 10 ganglia equivalents per well. To enhance neuron survival, nodose ganglia were enzymatically dissociated using neutral protease. Estimated survival at 5 days was 20–30%, with 800–1200 surviving neurons per plated ganglion, and decreased slowly thereafter. Specific SP immunostaining was present in 10–20% of neurons, mostly of small diameter (18–22 μm). SP content was low for 5 days then rose progressively after 14 days to 80–150 pg per plated ganglion. The addition of nerve growth factor (NGF, 100 ng/ml) to the culture medium did not alter neuron survival. However, SP content was doubled in the presence of NGF, or fell rapidly to one-half control levels following its withdrawal: e.g. following 12 days in culture with NGF1185 ± 176pg/well vs NGF withdrawn day 8–12,592 ± 118pg/well, mean±S.D., P < 0.01. Somatostatin, present in one-sixth the amount of SP, was unaltered by NGF. In subsequent studies, plating of neurons onto previously dissociated rat atriacytes increased survival by 50% but did not alter SP content per surviving neurons. These studies demonstrate that SP is present in dissociated cultures of rat vagal sensory neurons; the quantities and estimated net synthesis rate correspond to previous observations in vivo. The studies also demonstrate that SP content but not neuron survival are regulated by NGF in nodose ganglion neurons. This model may prove valuable for the study of SP and other sensory neuropeptides in this important class of visceral afferent neurons.

The expression of membrane and secreted immunoglobulin during the in vitro differentiation of the murine B cell lymphoma CH12.

The expression of membrane and secreted IgM was analyzed during mitogen-induced differentiation of the murine B cell lymphoma CH12. To characterize the Ig genes used by CH12, the nucleotide sequences of the variable gene segments (V mu and V kappa) were determined. The expressed V mu gene segment belongs to the VHII NPb-related family. The D (FL16.1a) and J (JH2) segments are the same as those used by the NP-specific hybridoma B1-8. The V kappa used by CH12 is almost identical to those used by the oxazolone-specific hybridomas NQ5.89.4 and NQ7.7.1. Treatment with lipopolysaccharide (LPS) induces up to 80% of CH12 cells to secrete IgM within 48 hr of culture. The steady state levels of secreted mu (mu s) and kappa mRNA increase four to fivefold over this period in cells stimulated with LPS compared with unstimulated cells. The kinetics are similar for both mRNA and parallel the increase in IgM secretion. EL-4 supernatants induce comparable changes in m mu s and kappa transcript levels. The simultaneous increase in m mu s and kappa transcripts suggests that coordinate control of RNA levels is used to increase the synthesis of secretory IgM during differentiation. The level of mRNA encoding the membrane form of mu (mu m) remains constant in stimulated cells and increases slightly in unstimulated cells. While the net rates of synthesis of membrane-bound mu-chains remain similar during LPS stimulation, the level of surface IgM on secreting cells is reduced three to fivefold. These observations suggest that the level of surface IgM expression during differentiation of CH12 is controlled largely by post-translational mechanisms. Our results demonstrate that the CH12 cell line regulates the expression of membrane and secreted IgM differently during its differentiation. The changes in IgM expression in CH12 parallel those occurring in normal B cells after mitogen or antigen challenge. Thus, the in vitro differentiation of CH12 is a good model for the analysis of late stages of B cell development.

Pyrimidine nucleotide metabolism in cardiac hypertrophy.

Cardiac hypertrophy was induced in rats either by isoprenaline treatment or by experimental abdominal aortic stenosis. In both models, the UTP content increased in the hypertrophying heart, while the ATP level remained depressed. The rate of net synthesis of uracil nucleotides, estimated by phosphate incorporation into alpha-phosphate groups, was largely increased during the first stage of hypertrophy (X 3.6 and 2.6, respectively). At identical stages of hypertrophy, the rate of synthesis of ribosomal RNA, measured after continuous intracardiac infusion of 3H-uridine, was increased by 1.6 and 2.5 times. A comparison of the changes in time course of the levels of UTP and RNA and their turnover suggests that the metabolism of uracil nucleotides and that of ribosomal RNA are closely interrelated. The increase in the rate of synthesis of pyrimidine is interpreted as an adaptational change in nucleotide metabolism in response to the increased requirement of precursors for RNA synthesis. The possible limiting role of pyrimidine nucleotides in the hypertrophic process is discussed.

Regulatory aspects of the glutamylation of methotrexate in cultured hepatoma cells

The glutamylation of methotrexate has been evaluated in H35 hepatoma cells in vitro as a function of the conditions of culture. Glutamylation yields methotrexate polyglutamate with two to five additional glutamate residues and is a saturable process. The rate of glutamylation increases little above 10 μ m extracellular methotrexate which corresponds to an intracellular concentration of approximately 4 μ m. The rate of glutamylation measured over a 6-h period was stimulated by a reduction in cellular folates and prior incubation of the cells with insulin. Glutamylation was also more rapid in dividing cultures than in confluent cells. The combination of insulin inclusion and folate reduction, which was additive, caused approximately a fourfold increase in the rate of glutamylation over control cells under the conditions tested. The maximal rate of methotrexate glutamylation, which was 100 nmol/g/h, occurred in folate-depleted, insulin-supplemented cells. Supplementing folate-depleted cells with reduced folate coenzymes caused the glutamylation to be reduced by more than 90%. The turnover of methotrexate polyglutamates in cells saturated with these derivatives occurred at approximately one-half the rate of net synthesis and was stimulated to nearly the same extent by folate depletion and insulin. In addition to showing that folates can modify the rates of methotrexate polyglutamate formation, data are presented suggesting that methotrexate polyglutamates can regulate their own synthesis. The consequences of the formation of these retained forms of methotrexate in H35 hepatoma cells ( M. Balinska, J. Galivan, and J. K. Coward (1981) Cancer Res. 41, 2751–2756 ) and the effects of potential regulators of this process are discussed in terms of the glutamylation of folates in the cells and the chemotherapeutic effects of antifolates.