Increased Incorporation Of Label Research Articles

Contributions of glycogen to astrocytic energetics during brain activation.

Glycogen is the major store of glucose in brain and is mainly in astrocytes. Brain glycogen levels in unstimulated, carefully-handled rats are 10-12 μmol/g, and assuming that astrocytes account for half the brain mass, astrocytic glycogen content is twice as high. Glycogen turnover is slow under basal conditions, but it is mobilized during activation. There is no net increase in incorporation of label from glucose during activation, whereas label release from pre-labeled glycogen exceeds net glycogen consumption, which increases during stronger stimuli. Because glycogen level is restored by non-oxidative metabolism, astrocytes can influence the global ratio of oxygen to glucose utilization. Compensatory increases in utilization of blood glucose during inhibition of glycogen phosphorylase are large and approximate glycogenolysis rates during sensory stimulation. In contrast, glycogenolysis rates during hypoglycemia are low due to continued glucose delivery and oxidation of endogenous substrates; rates that preserve neuronal function in the absence of glucose are also low, probably due to metabolite oxidation. Modeling studies predict that glycogenolysis maintains a high level of glucose-6-phosphate in astrocytes to maintain feedback inhibition of hexokinase, thereby diverting glucose for use by neurons. The fate of glycogen carbon in vivo is not known, but lactate efflux from brain best accounts for the major metabolic characteristics during activation of living brain. Substantial shuttling coupled with oxidation of glycogen-derived lactate is inconsistent with available evidence. Glycogen has important roles in astrocytic energetics, including glucose sparing, control of extracellular K(+) level, oxidative stress management, and memory consolidation; it is a multi-functional compound.

Changes in microtubule turnover accompany synaptic plasticity and memory formation in response to contextual fear conditioning in mice

Synaptic plasticity plays a crucial role in learning, memory, and cognitive disorders. Cytoskeletal reorganization underlies neuronal synaptic plasticity, but little is known about the regulation of cytoskeletal dynamics in living animals. We used stable isotope labeling to measure the turnover of tubulin in defined microtubule (MT) populations in murine brain. Neuronal MTs generally exhibited low turnover rates in vivo. Basal turnover was highest in tau-associated MTs, intermediate in microtubule-associated protein 2 (MAP2)–associated MTs, and lowest in cold-stable MTs. Labeling of MTs in mature neurons in cell culture yielded similar turnover results. Intracerebroventricular glutamate injection stimulated, via N-methyl-d-aspartic acid receptors, label incorporation (turnover) in cold-stable, tau-associated, and MAP2-associated MTs, the last of which was shown to be dependent on cyclic adenosine-3′, 5′-monophosphorothioate–protein kinase A. Contextual fear conditioning, a hippocampus-mediated form of memory formation, was accompanied by increased turnover of hippocampal MAP2-associated and cold-stable MTs. Treatment with the MT-depolymerizing drug nocodazole reversed the conditioning-induced increase in label incorporation in MAP2-associated MTs, reduced dendritic spine density, and impaired memory formation. The effects of nocodazole on MT turnover were prevented by the MT-stabilizing agent Taxol (Sigma-Aldrich, St. Louis, MO, USA) and by brain-derived nerve growth factor, both of which also restored dendritic spine density and memory formation in this model. In conclusion, these results suggest that changes in hippocampal MT turnover are required for, and are a biomarker of, the synaptic plasticity that is involved in memory formation.

Stirred vessel cultures of rat brain cells aggregates: Characterization of major metabolic pathways and cell population dynamics

We report a study on neural metabolism of long-term three-dimensional cultures of rat embryonic brain cells in stirred vessels. Our experimental setup was optimized to keep viable aggregate cultures with neuronal maintenance for up to 44 days. Results show that aggregate size and shape could be hydrodynamically controlled depending on the impeller design, avoiding necrotic centers or significant losses in cell viability. Aggregates were composed mainly of neurons until day 16, whereas an effective growth of the glial population was observed after day 21. Cell metabolic status was evaluated by quantification of several metabolites in the culture medium; amino acid metabolism was used as a marker of metabolic interrelationships between neural cell types. Furthermore, (13)C-NMR spectroscopy was used on day 31 to explore specific metabolic pathways: incubation with [1-(13)C]glucose for 45 hr produced an increase in label incorporation in extracellular alanine, lactate, and glutamine, reflecting mainly astrocytic metabolism. The contribution of anaplerotic vs. oxidative pathways for glutamine synthesis was determined: a 92% reduction in the pyruvate carboxylase flux during the first 41 hr of incubation suggested a decrease in the need for replacing tricarboxylic acid cycle intermediates. We believe that our data corroborate the aggregating cultures as an attractive system to analyze brain cell metabolism being a valuable tool to model metabolic fluxes for in vitro brain diseases.

In Vivo Measurement of Microtubule Dynamics Using Stable Isotope Labeling with Heavy Water: EFFECT OF TAXANES

Microtubules are dynamic polymers with central roles in the mitotic checkpoint, mitotic spindle assembly, and chromosome segregation. Agents that block mitotic progression and cell proliferation by interfering with microtubule dynamics (microtubule-targeted tubulin-polymerizing agents (MTPAs)) are powerful antitumor agents. Effects of MTPAs (e.g. paclitaxel) on microtubule dynamics have not yet been directly demonstrated in intact animals, however. Here we describe a method that measures microtubule dynamics as an exchange of tubulin dimers into microtubules in vivo. The incorporation of deuterium ((2)H(2)) from heavy water ((2)H(2)O) into tubulin dimers and polymers is measured by gas chromatography/mass spectrometry. In cultured human lung and breast cancer cell lines, or in tumors implanted into nude mice, tubulin dimers and polymerized microtubules exhibited nearly identical label incorporation rates, reflecting their rapid exchange. Administration of paclitaxel during 24 h of (2)H(2)O labeling in vivo reduced (2)H labeling in polymers while increasing (2)H in dimers, indicating diminished flux of dimers into polymers (i.e. inhibition of microtubule dynamic equilibrium). In vivo inhibition of microtubule dynamics was dose-dependent and correlated with inhibition of DNA replication, a stable isotopic measure of tumor cell growth. In contrast, microtubule polymers from sciatic nerve of untreated mice were not in dynamic equilibrium with tubulin dimers, and paclitaxel increased label incorporation into polymers. Our results directly demonstrate altered microtubule dynamics as an important action of MTPAs in vivo. This sensitive and quantitative in vivo assay of microtubule dynamics may prove useful for pre-clinical and clinical development of the next generation of MTPAs as anticancer drugs.

276 Phase I trial of low dose interferon-alpha (IFN), thalidomide with gemcitabine and capecitabine in patients with progressive metastatic renal cell carcinoma (RCC)

Limited information is available concerning the molecular and cellular mechanisms that regulate expression of insulin-like growth factor-I (IGF-I) binding proteins (IGFBPs) in bovine mammary epithelial cells. Here, we report on the autocrine mechanisms of action of IGF-I and hormonal regulation of expression of IGFBPs in bovine mammary epithelial MD-IGF-I cells which express recombinant IGF-I under the control of the glucocorticoid-inducible mouse mammary tumor virus-long terminal repeat (MMTV-LTR). Levels of IGFBP-3 mRNA and secretion of IGFBP-3 by MD-IGF-I cells were stimulated by IGF-I, insulin (INS), and IGF-I analogs but not prolactin (PRL). Conversely, parental MAC-T cells expressed little IGF-I and secreted primarily IGFBP-2 (29–32 kDa) in response to stimulation with INS, dexamethasone (DEX), or IGF-I analogs. Secretion of recombinant IGF-I caused a 26.5-fold increase in secretion of IGFBP-3, as measured by densitometric analysis of ligand blots, which was associated with a 1.7-fold increase in total DNA. Conditioned media (CM) from MD-IGF-I cells induced with DEX stimulated a 2.8-fold increase in [3H]thymidine incorporation into DNA of parental MAC-T cells, compared with uninduced cells. Moreover, inclusion of exogenous IGF-I with CM from MD-IGF-I cells triggered an additional 3.0-fold increase in label incorporation, but only a 1.6-fold increase in the presence of IGFBP-2-containing media conditioned by MAC-T cells. Des(l-3)IGF-I added to CM from both MAC-T and MD-IGF-I cells respectively, stimulated a 10.2 and 6.9-fold increase in [3H]thymidine incorporation into DNA of MAC-T cells. We suggest that expression of IGF-I-induced IGFBP-3 is an important component of an autocrine loop which potentiates the local mitogenic actions of IGF-I in bovine mammary epithelial cells.

Farnesyl Pyrophosphate Synthase Is the Molecular Target of Nitrogen-Containing Bisphosphonates

Bisphosphonates (Bps), inhibitors of osteoclastic bone resorption, are used in the treatment of skeletal disorders. Recent evidence indicated that farnesyl pyrophosphate (FPP) synthase and/or isopentenyl pyrophosphate (IPP) isomerase is the intracellular target(s) of bisphosphonate action. To examine which enzyme is specifically affected, we determined the effect of different Bps on incorporation of [14C]mevalonate (MVA), [14C]IPP, and [14C]dimethylallyl pyrophosphate (DMAPP) into polyisoprenyl pyrophosphates in a homogenate of bovine brain. HPLC analysis revealed that the three intermediates were incorporated into FPP and geranylgeranyl pyrophosphate (GGPP). In contrast to clodronate, the nitrogen-containing Bps (NBps), alendronate, risedronate, olpadronate, and ibandronate, completely blocked FPP and GGPP formation and induced in incubations with [14C]MVA a 3- to 5-fold increase in incorporation of label into IPP and/or DMAPP. Using a method that could distinguish DMAPP from IPP on basis of their difference in stability in acid, we found that none of the NBps affected the conversion of [14C]IPP into DMAPP, catalyzed by IPP isomerase, excluding this enzyme as target of NBp action. On the basis of these and our previous findings, we conclude that none of the enzymes up- or downstream of FPP synthase are affected by NBps, and FPP synthase is, therefore, the exclusive molecular target of NBp action.

Regulation of FFA by the acyltransferase pathway in focal cerebral ischemia-reperfusion.

Cerebral insult is associated with a rapid increase in free fatty acids (FFA) and arachidonic acid release has been linked to the increase in eicosanoid biosynthesis. In transient focal cerebral ischemia induced by middle cerebral artery (MCA) occlusion, there is an inverse relationship between the increase in FFA and the decrease in ATP, both during the ischemia period and at later time periods after reperfusion. In this study, the focal cerebral ischemia model was used to examine incorporation of [14C]arachidonic acid into the glycerolipids in rat MCA cortex at different reperfusion times after a 60 min ischemia. The label was injected intracerebrally into left and right MCA cortex 1 hr prior to decapitation. Labeled arachidonic acid was incorporated into phosphatidylcholine, phosphatidylethanolamine and neutral glycerides. With increasing time (4-16 hr) after a 60 min ischemia, an inhibition of labeled arachidonate uptake could be found in the right ischemic MCA cortex, whereas the distribution of radioactivity among the major phospholipids was not altered. When compared to labeled PC, there was a 3-4 fold increase in incorporation of label into phosphatidic acid and triacylglycerols (TG) in the right MCA cortex, suggesting of an increase in de novo biosynthesis of TG. In an in vitro assay system, synaptosomal membranes isolated from MCA cortex 8 and 16 hr after a 60 min ischemia showed a significant decrease in arachidonoyl transfer to lysophospholipids, due mainly to a decrease in lysophospholipid:acylCoA acyltransferase activity. Assay of phospholipase A2 activity with both syaptosomes and cytosol, however, did not show differences between left and right MCA cortex or with time after reperfusion. These results suggest that besides ATP availability, the decrease in acyltransferase activity may also contribute to the increase in FFA in cerebral ischemia-reperfusion.

IGF-I-Induced IGFBP-3 potentiates the mitogenic actions of IGF-I in mammary epithelial MD-IGF-I cells

Limited information is available concerning the molecular and cellular mechanisms that regulate expression of insulin-like growth factor-I (IGF-I) binding proteins (IGFBPs) in bovine mammary epithelial cells. Here, we report on the autocrine mechanisms of action of IGF-I and hormonal regulation of expression of IGFBPs in bovine mammary epithelial MD-IGF-I cells which express recombinant IGF-I under the control of the glucocorticoid-inducible mouse mammary tumor virus-long terminal repeat (MMTV-LTR). Levels of IGFBP-3 mRNA and secretion of IGFBP-3 by MD-IGF-I cells were stimulated by IGF-I, insulin (INS), and IGF-I analogs but not prolactin (PRL). Conversely, parental MAC-T cells expressed little IGF-I and secreted primarily IGFBP-2 (29–32 kDa) in response to stimulation with INS, dexamethasone (DEX), or IGF-I analogs. Secretion of recombinant IGF-I caused a 26.5-fold increase in secretion of IGFBP-3, as measured by densitometric analysis of ligand blots, which was associated with a 1.7-fold increase in total DNA. Conditioned media (CM) from MD-IGF-I cells induced with DEX stimulated a 2.8-fold increase in [ 3H]thymidine incorporation into DNA of parental MAC-T cells, compared with uninduced cells. Moreover, inclusion of exogenous IGF-I with CM from MD-IGF-I cells triggered an additional 3.0-fold increase in label incorporation, but only a 1.6-fold increase in the presence of IGFBP-2-containing media conditioned by MAC-T cells. Des(l-3)IGF-I added to CM from both MAC-T and MD-IGF-I cells respectively, stimulated a 10.2 and 6.9-fold increase in [ 3H]thymidine incorporation into DNA of MAC-T cells. We suggest that expression of IGF-I-induced IGFBP-3 is an important component of an autocrine loop which potentiates the local mitogenic actions of IGF-I in bovine mammary epithelial cells.

Isolation and in vitro translation of messenger RNA from the American cockroach.

Total RNA was extracted from the whole body of American cockroach (Periplaneta americana) using chaotropic salt guanidine isothiocyanate in the presence of 2-mercaptoethanol (2-ME). Polyadenylated mRNA was isolated by oligothymidylic acid-cellulose chromatography and mRNA was translated using a rabbit reticulocyte lysate system. The translation, as judged by the incorporation of 35S-methionine, was obtained with poly(A)+ RNA, where an approximately 9.5-fold increase in label incorporation over control was achieved. Analysis of translation products by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) in combination with autoradiography showed that many proteins with apparent molecular weights ranging from 12 to 200 kD were synthesized, and no labelled proteins were found with negative RNA control and poly(A)- RNA. Immunoprecipitation studies performed using polyclonal and monoclonal antibodies revealed that synthesized proteins of MW 90, 78, 72, 49, 45, and 26 kD corresponded with previously identified principal and major allergens of American cockroach from our laboratory. In addition, the allergenicity of the translation mixtures was also confirmed by fluoroallergosorbent test (FAST) inhibition studies with IgE antibodies of human reaginic serum pool.

Effects of brefeldin A on sphingomyelin transport and lipid synthesis in BHK21 cells.

1. Addition of brefeldin A (BFA) to BHK cells incubated for 4 h with [3H]acetate led to a 3-4-fold increase in incorporation of label into sphingomyelin, monoglucosylceramide and cholesterol ester compared with untreated controls. There was a similar increase in incorporation of [3H]choline into sphingomyelin. The level of cholesterol ester increased 3-fold when BFA was added to cells labelled to equilibrium with [3H]acetate, but no statistically significant changes in the levels of other lipids were seen. 2. BFA appeared to act by diverting incorporation of acetate into sphingolipids and cholesterol ester at the expense of phosphatidylcholine (decreased by up to 15%), cholesterol (decreased by 30-40%) and triacylglycerol (decreased by 35-50%). 3. Forskolin (100 microM) prevented the changes in labelling induced by 0.25 micrograms of BFA/ml, but in the presence of 1 micrograms of BFA/ml it had no effect on sphingomyelin and triacylglycerol labelling and only partly blocked the effects of BFA on labelling of cholesterol and cholesterol ester. 4. None of the labelled sphingomyelin was degraded in BFA-treated cells which were subsequently exposed to an extracellular sphingomyelinase, showing that all the newly synthesized sphingomyelin remained inside the cells. Determinations of phospholipid phosphorus in unlabelled cells confirmed that, in the presence of BFA, no newly synthesized sphingomyelin was able to reach the cell surface, supporting the idea that sphingomyelin normally depends on vesicular transport for its passage to the plasma membrane. 5. The results are consistent with the hypothesis that cholesterol synthesis and esterification processes in BHK cells are sensitive to the plasma-membrane deficit of sphingomyelin caused by BFA.

Lipopolysaccharide (LPS) Alters Phosphatidylcholine Metabolism in Elicited Peritoneal Macrophages

We investigated the effects of LPS on mouse peritoneal macrophage phospholipids using radiolabeled precursors. LPS (200 ng/ml) stimulated incorporation of [32P] into all classes of phospholipids within 0.5 hr, and after 2 hr the increase was 60% greater than controls. Separation of the phospholipid classes by thin-layer chromatography revealed a selective increase in incorporation of label into phosphatidylcholine (PC) (90% increase compared to approximately 50% in the other phospholipids). In macrophages labeled with [3H]-choline, LPS stimulated both the incorporation of label into PC and the release of incorporated label into the medium. The time dependencies of stimulated [3H] release and [32P] incorporation were similar. These data are consistent with the hypothesis that LPS activates macrophages via a PC-specific phospholipase-dependent mechanism.

DNA repair following incorporation of 5-fluorouracil into DNA of mouse bone marrow cells.

5-Fluorouracil (FUra) was previously demonstrated to be incorporated into DNA at cytotoxic concentrations in mouse bone marrow cells. Subsequently, we showed that under these conditions FUra exhibited a time-dependent removal from DNA accompanied by a decrease in DNA strand length. In the present study we utilized hydroxyurea to inhibit semiconservative DNA synthesis while monitoring DNA repair by measuring the incorporation of [3H]dThd into double-stranded DNA (DNAds), which can be separated from DNA at the replication fork (DNAss) by benzoylated-naphthoylated-DEAE cellulose. Under these conditions we assessed DNA repair in cells that had previously been exposed for 1 h to varying cytotoxic concentrations of FUra. The results demonstrate an increase in labelling of DNAds with increasing FUra concentrations, with no appreciable increase in incorporation of label into DNAss. In conclusion, this study demonstrates that DNA repair occurs following incorporation of FUra. The failure to repair DNA damage at higher FUra concentrations may have a role in the cytotoxicity of this drug.

Influence of light quality on the ribosomal RNA levels inWolffia arrhiza: Comparison of phosphate and magnesium limited chemostat populations

Wolffia arrhiza (L.) Wimm. was grown axenically in the chemostat under white luminescent light (photon fluence rate 23 ujnol m-2 s-1) and phosphate or magnesium limitation (0.075 and 0.01 jxmol 1-1, respectively). Aliquots (1 g fresh mass) were taken from the continuous cultures and were irradiated for 1 h with either white light (control) or monochromatic blue (453 nm) or red (654 nm) light. The amount of [5-3H]-uridine incorporated into cytosolic and chloroplastic rRNAs during these exposures was estimated and following results were obtained: In phosphate limited plants rod light considerably reduced and blue light slightly increased label incorporation as compared with the control. Moreover, in red light, chloroplast incorporation is relatively more slowed down than that in the cytosolic compartment (34 % as compared to 59 % of the control). In blue light the enhancement is approximately equal in both compartments. In magnesium limited plants incorporation under both blue and red light is moderately slower as compared with the control. In both cases also the retardation is slightly greater in the chloroplast than in the cytoplasm. The results suggest that rRNA metabolism is controlled by light quality as well as by mineral nutrition.

Effects of Volatile Fatty Acids on Propionate Metabolism and Gluconeogenesis in Caprine Hepatocytes

Isolated caprine hepatocytes were incubated with fatty acids of various chain lengths. Short-chain fatty acids effects on rates of gluconeogenesis and oxidation from [2-14C]propionate were determined. Additions of glucose (2.5mM) had no effect on hepatic [2-14C]propionate metabolism in the presence and absence of amino acids. A complete mixture of amino acids increased label incorporation from [2-14C]propionate into [14C]glucose by 22%. Butyrate inhibited [2-14C]propionate metabolism and increased the apparent Michaelis constant for [2-14C]propionate incorporation into [14C]glucose from 2.4±1.5 to 5.6±.9mM. Butyrate's effects on propionate were similar in the presence and absence of L-carnitine (1mM). Isobutyrate, 2-methylbutyrate, and valerate (1.25mM) had no effect on [14C]glucose production but decreased 14CO2 production to 57, 61, and 54% of the control [2-14C]propionate (1.25mM). This inhibition on 14CO2 production was not competitive. Isovalerate had no effect on either [2-14C] propionate incorporation into glucose or CO2. An increase in ratio of [14C]glucose to 14CO2 from [2-14C]propionate demonstrated that short-chain fatty acids other than butyrate do not inhibit gluconeogenesis from propionate. In addition, fatty acids that generate a net synthesis of intracellular oxaloacetate may partition propionate carbons toward gluconeogenic rather than oxidative pathways in goat hepatocytes.

Regulation of low density lipoprotein receptor synthesis in cultured luteinized human granulosa cells by human chorionic gonadotropin and 8-bromo-cyclic AMP.

We investigated the regulation of synthesis of low density lipoprotein (LDL) receptor in cultured luteinized human granulosa cells using a monoclonal antibody recognizing the human LDL receptor (IgG-C7). Cells cultured under serum-free conditions were treated with human chorionic gonadotropin (hCG) or 8-bromo-cAMP alone or in combination with aminoglutethimide (to block conversion of cholesterol to steroid hormones) and 5-cholesten-3 beta, 25-diol (25-hydroxycholesterol, a potent suppressor of LDL receptor expression in human fibroblasts) and pulse-labeled with [35S]methionine. A labeled protein immunoisolated with IgG-C7 was identified as the mature LDL receptor in 7.5% sodium dodecyl sulfate-polyacrylamide gels on the basis of an apparent molecular mass of 160 kDa, absence of the protein from immunoisolates prepared with a monoclonal antibody against an irrelevant antigen, and an apparent decrease in molecular weight of the mature receptor upon treatment with neuraminidase or electrophoresis under nonreducing conditions. hCG and 8-bromo-cAMP consistently increased the incorporation of radioactivity into the mature LDL receptor by 2-6-fold. The effect of hCG on LDL receptor synthesis was observed with as little as 10 mIU of hCG/ml and was apparent within 2 h of addition of the hormone. A combination of 25-hydroxycholesterol and aminoglutethimide resulted in a 60% suppression of label incorporation into mature LDL receptor compared to untreated cells. This would suggest some regulation of LDL receptor synthesis by negative feedback of sterol. However, both hCG and 8-bromo-cAMP increased label incorporation into the LDL receptor in the face of these agents. We conclude that in human granulosa cells, hCG, through the intermediacy of cAMP, rapidly increases LDL receptor synthesis by a mechanism which is, at least in part, independent of alterations in cellular cholesterol balance.

The subfornical organ: Biochemical and neuroendocrine comparisons with the hypothalamo-neurohypophysial system

Biochemical, cytochemical and immunological methods were used to compare the metabolic and neuroendocrine properties of the subfornical organ (SFO) with the hypothalamo-neurohypophysial system (HNS) in the rat. The SFO resembles the HNS in that both have (a) increased label incorporation into RNA during dehydration; (b) an intense reaction for glucose-6-phosphate dehydrogenase; (c) NADPH-diaphorase and the Type I pathway for hydrogen utilization from NADPH-diaphorase and the Type I pathway for hydrogen utilization from NADPH, presumably as part of the mixed-function oxidase system for the metabolism of endogenous substrates and xenobiotics; (d) immunoreactive vasopressin and oxytocin. Gel filtration of extracts of the SFO area using Sephadex G-25 chromatography resulted in immunoreactive peaks for both AVP and OT which were similar to synthetic hormones. One other fraction in the SFO extract, containing a substance(s) of higher molecular weight than AVP, was detected using the chromatography resulted in immunoreactive peaks for both AVP and OT which were similar to synthetic hormones. One antiserum for AVP. The concentration of immunoreactive AVP in the SFO area was increased after colchicine, decreased by hypophysectomy, and unaltered by: (a) infusion (4.6 pg/min for 3 hr) or injection (1 or 6 ng) of AVP into the lateral cerebroventricle; (b) dehydration; (c) renin administered intracerebroventricularly; (d) pinealectomy; or (e) hypertension in the spontaneously hypertensive rat. In conclusion, cells in the SFO have specialized metabolic and neuroendocrine properties similar to the HNS. It can be inferred from these biochemical specializations that the SFO has metabolic and secretory activities.

Biochemical mechanisms of the antileishmanial activity of sodium stibogluconate

Pentavalent antimonial agents such as sodium stibogluconate (Pentostam; Burroughs Wellcome Co., London, United Kingdom) are the drugs of choice for the treatment of leishmaniasis, but their biochemical mechanisms of action are virtually unknown. The viability of Leishmania mexicana (WR 227) promastigotes and amastigotes was decreased 40 to 61% by a 4-h exposure to 500 micrograms of Sb (in the form of stibogluconate) per ml. Such exposure also resulted in a 51 to 65% decrease in incorporation of label into DNA, RNA, and protein; a 56 to 65% decrease in incorporation of label into purine nucleoside triphosphate; and a 34 to 60% increase in incorporation of label into purine nucleoside monophosphate and diphosphate. It is postulated that the apparent decrease in ATP and GTP synthesis from ADP and GDP contributes to decreased macromolecular synthesis and to decreased Leishmania viability. Further experiments suggested that inhibition of glycolysis and the citric acid cycle may partially explain the inability to phosphorylate ADP.

The effect of X-irradiation on membrane lipids of lymphosarcoma cells in vivo and in vitro.

Phospholipids of the membranes of spleen lymphosarcoma cells were radioactively labelled with the radioresistant fatty acid palmitic acid (16:0) and the radiosensitive fatty acid arachidonic acid (20:4). The effect of irradiation on the specific radioactivity of the phospholipids was studied. For the in vivo experiments trace amounts of radioactive palmitate or arachidonate were injected intraperitoneally into lymphosarcoma-bearing mice. Incorporation of label into the lipids of the tumorous spleen cells was monitored in control animals and in mice that were whole body irradiated after injection of the label. In both groups of animals the label was detected in the blood within minutes after injection and was found to be efficiently incorporated into the phospholipids of the tumor cells. In the irradiated animals a marked transient increase in label incorporation was observed as compared to control animals. The radiation effect was observed in the lipids of the total homogenate, purified nuclei, the mitochondriallysosomal fraction and in the microsomal fraction. Most experiments were performed with nuclei, which are known for their high radiosensitivity. The levels of label incorporation for palmitate and arachidonate were increased to the same extents and found to be dose dependent. For a dose of 5 Gy the increased label incorporation started immediately after irradiation and lasted for a period of about 50 minutes. The increase in label incorporation into the phospholipids was preceded by an increase in the concentration of fatty acids in the cytosol of the tumors. Our experiments point to the occurrence of a transient increase in the flux of fatty acids through the plasma membrane as a result of irradiation and suggest that under normal physiological conditions fatty acid uptake through the plasma membrane is the rate-limiting step in the incorporation of acyl groups into the phospholipids. Experiments with isolated tumor cells also showed an increased incorporation of fatty acids into the phospholipids after irradiation. Again the incorporation patterns of both types of fatty acids (16:0) and (20:4) were very similar. A hypotonic treatment of the cells also resulted in a similar increase in fatty acid incorporation as irradiation did; the effects of hypotonic treatment and irradiation were not additive.

The regulation of yolk polypeptide synthesis in Drosophila ovaries and fat body by 20-hydroxyecdysone and a juvenile hormone analog

In adult female Drosophila melanogaster an increase in the synthesis and secretion of three yolk polypeptides (YPs) occurs during the first 24 hr after eclosion. During organ culture, these same polypeptides are synthesized and secreted into the medium by both fat body and ovaries. Two hormones, 20-hydroxyecdysone (20-HE) and a juvenile hormone analog (ZR-515) stimulate synthesis and secretion of YPs into the hemolymph of isolated female abdomens. The present experiments were undertaken to compare synthesis of YPs in normal females with YP synthesis in preparations deprived of anterior endocrine glands, and to find which hormone stimulates synthesis in the different organs. Separation of hemolymph proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that at eclosion incorporation of [ 35S]methionine into YP1 and YP2 was low and was barely detectable in YP3. Over the next 24 hr the rate of label incorporation increased for all the YPs. Isolation of female abdomens at eclosion prevented this increase in label incorporation but did not entirely abolish YP synthesis. Application of either ZR-515 or 20-HE to isolated abdomens stimulated up to ninefold label incorporation into three polypeptides which comigrated with YPs from normal vitellogenic females. The response of isolated abdomens to ZR-515 or 20-HE was first detectable between 90 and 135 min after hormone application. The stimulated bands were confirmed to be YPs by a comparison of peptide digests of each of the three labeled polypeptides with those of the yolk polypeptides from intact vitellogenic females. The hypothesis that the two hormones might act on different organs was tested by treating isolated female abdomens with various concentrations of either ZR-515 or 20-HE and then culturing the stimulated organ in vitro with [ 35S]methionine. The fat body responded to both hormones by synthesizing and secreting into the culture medium polypeptides which comigrated with the YPs found in hemolymph, whereas the ovary produced similar polypeptides only after ZR-515. These secreted polypeptides were confirmed to be YPs by repeating the experiment using organs from heterozygotes for both YP2 and YP3 electrophoretic variants. Such organs synthesized five polypeptides which comigrated with the corresponding yolk polypeptides. These findings are discussed in relation to a hypothesis for the action of the two hormones.

Increased cellular proliferation in adipose tissue of adult rats fed a high-fat diet.

The feeding of a high-fat diet to adult rats was shown to increase the incorporation of [3H]thymidine into DNA of the adipocyte and stromal fractions. After only 2 days on a high-fat diet there was a marked increase in the incorporation of label. When a 2-week period was interposed between [3H]thymidine administration and determination of DNA specific activity, the greatest increase in incorporation of label was found after 1 week on the diet, when incorporation increased 6-fold or more in both adipocytes and stroma and subsequently decreased to stabilize at a level two or three times that of chow-fed rats in the adipocyte fraction. Rats labeled when young and later placed on a high-fat diet showed a decrease in DNA specific activity in both adipocytes and stroma, confirming that cellular proliferation had occurred in both fractions. The specific activities of both stromal and adipocyte DNA were very similar at all time points studied. An attempt to increase the difference in specific activities by waiting many weeks after [3H]thymidine injection before isolating DNA was not successful. This may be because the total amount of DNA in the stromal and adipocyte fractions increases in parallel on the diet. The significance of these findings in terms of the normal turnover of adipose tissue DNA and the responsiveness to diet is discussed.