Light regulation of sink metabolism in tomato fruit : I. Growth and sugar accumulation

Light/dark effects on growth and sugar accumulation in tomato (Lycopersicon esculentum) fruit during early development were studied on intact plants (in vivo) and in tissue culture (in vitro). Through the use of an in vitro culture of tomato fruit, it was possible to investigate the direct effects of light on sink metabolism by eliminating the source tissue. Similar growth patterns were found in vivo and in vitro. Fruit growth in different sugars indicated that sucrose was the best source of carbon for in vitro fruit growth. Fruit growth increased as sucrose concentration increased up to 8%. Darkening the fruit decreased fruit dry weight about 40% in vivo and in vitro. The differences in the CO₂ exchange rate between light and dark grown fruit indicated that light stimulation of fruit growth was due to mechanisms other than photosynthesis. Supporting this conclusion was the fact that light intensities ranging from 40 to 160 micromoles per square meter per second had no significant influence on fruit growth, and light did not increase growth of fruit cultured with glucose or fructose as a carbon source. However, light stimulated fruit growth significantly when sucrose was used as the carbon source. Light-grown fruit took up 30% more sucrose from the same source and accumulated almost twice as much hexose and starch as dark-grown fruit. A possible expansion of an additional sink for carbon by light stimulation of starch synthesis during early development will be discussed.     

Discovery of novel glitazones incorporated with phenylalanine and tyrosine: Synthesis,antidiabetic activity and structure–activity relationships

We report a series of new glitazones incorporated with phenylalanine and tyrosine. All the compounds were tested for their in vitro glucose uptake activity using rat-hemidiaphragm, both in presence and absence of insulin. Six of the most active compounds from the in vitro screening were taken forward for their in vivo triglyceride and glucose lowering activity against dexamethazone induced hyperlipidemia and insulin resistance in Wistar rats. The liver samples of rats that received the most active compounds, 23 and 24, in the in vivo studies, were subjected to histopathological examination to assess their short term hepatotoxicity. The investigations on the in vitro glucose uptake, in vivo triglyceride and glucose lowering activity are described here along with the quantitative structure–activity relationships.     

Evidence for a functional ATP-citrate lyase:NADP-malate dehydrogenase pathway in bovine adipose tissue: Enzyme and metabolite levels

Activities of key lipogenic and glycolytic enzymes were determined in extracts of crude homogenates to elucidate the rate-limiting step(s) for lipogenesis from lactate and glucose in bovine subcutaneous adipose tissue. The enzymes ATP-citrate lyase, NADP-malate dehydrogenase, and pyruvate carboxylase were shown to have enough activity to account for the rates of in vitro lipogenesis from 10 mm lactate with or without 2 mm glucose. Glucose utilization for fatty acid synthesis appears to be limited by the low activities of key glycolytic enzymes, especially hexokinase. Attempts were also made to estimate enzyme activities in bovine subcutaneous adipose tissue being incubated in vitro by relating primary substrate levels to kinetic characteristics for the enzymes. ATP-citrate lyase was estimated to be operating at levels equivalent to the rates of lactate incorporation into fatty acids in the absence or presence of 2 mm glucose in the incubation media. Additionally, metabolite levels were measured in rapidly frozen samples of bovine subcutaneous adipose tissue to estimate the relative importance of key lipogenic enzymes in vivo. At the citrate and malate levels measured in vivo, ATP-citrate lyase would be operating at levels that approximate those estimated in vitro.     

Lac repressor-operator interaction. 8. Lactose is an anti-inducer of the lac operon

Lactose is shown to be an effective anti-inducer of the lac operon both in vivo and in vitro. When lactose is used as a carbon source, the synthesis of β-galaetosidase in Escheriahia coli is not fully induced. Moreover, lactose is able to partially inhibit induction by isopropyl-(β-d-thiogalactoside in strains synthesizing inactive as well as active β-galactosidase. These effects in vivo are not due to catabolite repression by the glucose derived from lactose. These in vivo results suggest that lactose is acting as an anti-inducer. This is confirmed in vitro by showing that lactose binds to the lac represser and stabilizes the represser-operator complex.     

AMPK Activation through Mitochondrial Regulation Results in Increased Substrate Oxidation and Improved Metabolic Parameters in Models of Diabetes

Modulation of mitochondrial function through inhibiting respiratory complex I activates a key sensor of cellular energy status, the 5''-AMP-activated protein kinase (AMPK). Activation of AMPK results in the mobilization of nutrient uptake and catabolism for mitochondrial ATP generation to restore energy homeostasis. How these nutrient pathways are affected in the presence of a potent modulator of mitochondrial function and the role of AMPK activation in these effects remain unclear. We have identified a molecule, named R419, that activates AMPK in vitro via complex I inhibition at much lower concentrations than metformin (IC₅₀ 100 nM vs 27 mM, respectively). R419 potently increased myocyte glucose uptake that was dependent on AMPK activation, while its ability to suppress hepatic glucose production in vitro was not. In addition, R419 treatment of mouse primary hepatocytes increased fatty acid oxidation and inhibited lipogenesis in an AMPK-dependent fashion. We have performed an extensive metabolic characterization of its effects in the db/db mouse diabetes model. In vivo metabolite profiling of R419-treated db/db mice showed a clear upregulation of fatty acid oxidation and catabolism of branched chain amino acids. Additionally, analyses performed using both ¹³C-palmitate and ¹³C-glucose tracers revealed that R419 induces complete oxidation of both glucose and palmitate to CO₂ in skeletal muscle, liver, and adipose tissue, confirming that the compound increases mitochondrial function in vivo. Taken together, our results show that R419 is a potent inhibitor of complex I and modulates mitochondrial function in vitro and in diabetic animals in vivo. R419 may serve as a valuable molecular tool for investigating the impact of modulating mitochondrial function on nutrient metabolism in multiple tissues and on glucose and lipid homeostasis in diabetic animal models.     

In vitro development of the strobilar stage of Mesocestoides corti

Thompson R. C. A., Jue Sue L. P. and Buckley S. J. 1982. In vitro development of the strobilar stage of Mesocestoides corti. International Journal for Parasitology12: 303–314. Sexually mature strobilated adults of Mesocestoides corti were grown consistently from undifferentiated tetrathyridia in vitro using a conventional diphasic culture system. Development (growth, strobilisation and maturation) was compared in vitro and in vivo. Although growth and strobilisation were comparable in vitro and in vivo, during the first 18 days, total length and numbers of proglottids decreased in vivo but continued to increase in vitro after day 18. Both male and female reproductive systems appeared to develop normally in vitro and self copulation was frequently observed in cultured worms. However, fully developed oncospheres were not produced in vitro.     

Role of Amino Acids and Vitamins in Nutrition of Mesophilic Methanococcus spp

In this study we found that autotrophic methanococci similar to Methanococcus maripaludis obtained up to 57% of their cellular carbon from exogenous amino acids. About 85% of the incorporation was into protein. Primarily nonpolar and basic amino acids and glycine were incorporated; only small amounts of acidic and some polar amino acids were taken up. An additional 10% of the incorporation was into the nucleic acid fraction. Because little ¹⁴CO₂ was formed from the ¹⁴C-amino acids, little metabolism of the amino acids occurred. Therefore the growth stimulation by amino acids was probably due to the sparing of anabolic energy requirements. Of the amino acids incorporated, only alanine was also a sole nitrogen source for these methanococci. In contrast, Methanococcus vannielii and “Methanococcus aeolicus” are autotrophic methanococci which did not incorporate amino acids and did not utilize alanine as a sole nitrogen source. Although glutamine served as a sole nitrogen source for the autotrophic methanococci and Methanococcus voltae, a heterotrophic methanococcus, growth was due to chemical deamination in the medium. M. voltae requires leucine and isoleucine for growth. However, these amino acids were not significant nitrogen sources, and alanine was not a sole nitrogen source for the growth of M. voltae. The branched-chain amino acids were not extensively metabolized by M. voltae. Pantoyl lactone and pantoic acid were readily incorporated by M. voltae. The intact vitamin pantothenate was neither stimulatory to growth nor incorporated. In conclusion, although amino acids and vitamins are nutritionally important to both autotrophic and heterotrophic methanococci, generally they are not subject to extensive catabolism.     

Cellular immunity in the mouse. V. Further studies on leukemia virus activation in allogeneic reactions of mice: stimulatory parameters.

The relationship between activation of thymic (T)-derived lymphocytes and mouse leukemia virus (MuLV) induction were studied in vivo and in vitro. The results indicate that there is no simple relationship between the severity of GVH, assayed by splenomegaly, alteration of T-cell reactivity in vitro, the activation of mouse leukemia viruses, and the subsequent development of lymphoma. Allogeneic stimulation either in vivo or in vitro is a potent activator of MuLV, as is the drug iododeoxyuridine. However, nonspecific T-cell mitogens such as PHA or Con-A, the drug cyclophosphamide, or specific antigenic stimulus such as sheep red blood cells after in vivo sensitization are not effective virus activators. The source of the cell supporting MuLV replication in vitro appears to be a theta-positive (T) lymphoblast.     

Oesophagostomum radiatum: Glucose metabolism of larvae grown in vitro and adults grown in vivo

Larval stages of Oesophagostomum radiatum grown in vitro and adults grown in vivo were incubated in complex media or in a simple salt solution containing radioactive glucose. Glucose disappearance and end product accumulation of third-stage larvae in a simple salt solution indicated that they excreted CO₂ and acetic, propionic, and lactic acids. Larvae in third molt, fourth stage, and adults all excreted CO₂, acetic, propionic, and lactic acids at twice the rate of third-stage larvae plus an additional product, methylbutyric acid. Carbon dioxide arose primarily from the 3 or 4 carbons of glucose. An anaerobic atmosphere (95% N₂:5% CO₂) had no apparent effect on metabolism. When incubation was done in complex media, isobutyric and 3-methylbutyric acids were seen as major excretion products (10 and 24%, respectively). However, these acids were quantitatively minor when incubations took place in simple salts-glucose medium (1 and 0–3%, respectively).     

Efficacy of an essential oil of Eugenia caryophyllata against Psoroptes cuniculi

The acaricidal activity of Eugenia caryophyllata essential oil was evaluated in vitro and in vivo on Psoroptes cuniculi, a mange mite. In vitro, different concentrations of the oil were tested and the observed mites mortality was compared with that observed in untreated and treated (Acacerulen R^®) controls. In vivo, six P. cuniculi infected rabbits were topically treated with the oil diluted at 2.5% and compared with untreated and treated control groups of six rabbits each. In vitro, up to the concentration of 0.10% the oil gave highly significant (P < 0.01) percentages of mite mortality respect to the untreated controls, but only up to 0.16% it showed the same efficacy of Acacerulen R^®. In vivo, the treatment with the essential oil cured all infested rabbits and no statistical differences were observed respect to the treated control group. The untreated rabbits remained infested.     

Studies of glucose metabolism in Hymenolepis diminuta using 13C nuclear magnetic resonance

The metabolism in vitro of U-¹³C-glucose and NaH¹³CO₃ by two strains of adult Hymenolepis diminuta, the ANU and UT strains, was examined using ¹³C n.m.r. spectroscopy. The incubation medium and perchlorate extracts from worms incubated in vitro with U-¹³C-glucose showed incorporation of significant quantities of label into the end products succinate, lactate and acetate, and also into glycogen. Similar experiments with NaH¹³CO₃ showed incorporation principally into succinate C-1,4, plus significant labelling also in lactate C-1. This shows that nutochondrial malate or pyruvate contributes to the cytosolic pyruvate pool in H. diminuta. The metabolism of U-¹³C-glucose was followed directly by incubating live worms directly in the spectrometer. Worms from 24 h-fasted hosts metabolised the added glucose completely during an experimental period of 2 h and incorporation of label was evident in the time course spectra. Parasites from fed hosts metabolised the added glucose more slowly. This work confirms the accepted routes of glucose metabolism in H. diminuta and demonstrates the utility of the n.m.r. technique in investigating the metabolism of parasites.     

Cinnamic acid exerts anti-diabetic activity by improving glucose tolerance in vivo and by stimulating insulin secretion in vitro

Although the anti-diabetic activity of cinnamic acid, a pure compound from cinnamon, has been reported but its mechanism(s) is not yet clear. The present study was designed to explore the possible mechanism(s) of anti-diabetic activity of cinnamic acid in in vitro and in vivo non-obese type 2 diabetic rats. Non-obese type 2 diabetes was developed by injecting 90 mg/kg streptozotocin in 2-day-old Wistar pups. Cinnamic acid and cinnamaldehyde were administered orally to diabetic rats for assessing acute blood glucose lowering effect and improvement of glucose tolerance. Additionally, insulin secretory activity of cinnamic acid and cinnamaldehyde was evaluated in isolated mice islets. Cinnamic acid, but not cinnamaldehyde, decreased blood glucose levels in diabetic rats in a time- and dose-dependent manner. Oral administration of cinnamic acid with 5 and 10 mg/kg doses to diabetic rats improved glucose tolerance in a dose-dependent manner. The improvement by 10 mg/kg cinnamic acid was comparable to that of standard drug glibenclamide (5 mg/kg). Further in vitro studies showed that cinnamaldehyde has little or no effect on glucose-stimulated insulin secretion; however, cinnamic acid significantly enhanced glucose-stimulated insulin secretion in isolated islets. In conclusion, it can be said that cinnamic acid exerts anti-diabetic activity by improving glucose tolerance in vivo and stimulating insulin secretion in vitro.     

Variability in azygospore production among Entomophaga maimaiga isolates

This study describes in vitro and in vivo azygospore production by nine isolates of Entomophaga maimaiga, a fungal pathogen of the gypsy moth, Lymantria dispar. The three E. maimaiga isolates that consistently produced azygospores in vitro were also strong producers of azygospores in vivo. However, two additional isolates that were strong azygospore producers in vivo did not produce azygospores in vitro. Isolates that produced azygospores in vitro produced both conidia and azygospores more frequently in vivo than isolates not producing azygospores in vitro. In vitro azygospore production varied over time as well as by isolate. After >2 years of cold storage, while three isolates continued in vitro azygospore production, three isolates no longer produced azygospores in vitro.     

The Fate of Acetic Acid during Glucose Co-Metabolism by the Spoilage Yeast Zygosaccharomyces bailii

Zygosaccharomyces bailii is one of the most widely represented spoilage yeast species, being able to metabolise acetic acid in the presence of glucose. To clarify whether simultaneous utilisation of the two substrates affects growth efficiency, we examined growth in single- and mixed-substrate cultures with glucose and acetic acid. Our findings indicate that the biomass yield in the first phase of growth is the result of the weighted sum of the respective biomass yields on single-substrate medium, supporting the conclusion that biomass yield on each substrate is not affected by the presence of the other at pH 3.0 and 5.0, at least for the substrate concentrations examined. In vivo ¹³C-NMR spectroscopy studies showed that the gluconeogenic pathway is not operational and that [2−¹³C]acetate is metabolised via the Krebs cycle leading to the production of glutamate labelled on C₂, C₃ and C₄. The incorporation of [U-¹⁴C]acetate in the cellular constituents resulted mainly in the labelling of the protein and lipid pools 51.5% and 31.5%, respectively. Overall, our data establish that glucose is metabolised primarily through the glycolytic pathway, and acetic acid is used as an additional source of acetyl-CoA both for lipid synthesis and the Krebs cycle. This study provides useful clues for the design of new strategies aimed at overcoming yeast spoilage in acidic, sugar-containing food environments. Moreover, the elucidation of the molecular basis underlying the resistance phenotype of Z. bailii to acetic acid will have a potential impact on the improvement of the performance of S. cerevisiae industrial strains often exposed to acetic acid stress conditions, such as in wine and bioethanol production.     

4-Hydroxy hexenal derived from dietary n-3 polyunsaturated fatty acids induces anti-oxidative enzyme heme oxygenase-1 in multiple organs

It has recently been reported that expression of heme oxygenase-1 (HO-1) plays a protective role against many diseases. Furthermore, n-3 polyunsaturated fatty acids (PUFAs) were shown to induce HO-1 expression in several cells in vitro, and in a few cases also in vivo. However, very few reports have demonstrated that n-3 PUFAs induce HO-1 in vivo.     

Engineering an Endothelialized Vascular Graft: A Rational Approach to Study Design in a Non-Human Primate Model

After many years of research, small diameter, synthetic vascular grafts still lack the necessary biologic integration to perform ideally in clinical settings. Endothelialization of vascular grafts has the potential to improve synthetic graft function, and endothelial outgrowth cells (EOCs) are a promising autologous cell source. Yet no work has established the link between endothelial cell functions and outcomes of implanted endothelialized grafts. This work utilized steady flow, oscillatory flow, and tumor necrosis factor stimulation to alter EOC phenotype and enable the formulation of a model to predict endothelialized graft performance. To accomplish this, EOC in vitro expression of coagulation and inflammatory markers was quantified. In parallel, in non-human primate (baboon) models, the platelet and fibrinogen accumulation on endothelialized grafts were quantified in an ex vivo shunt, or the tissue ingrowth on implanted grafts were characterized after 1mth. Oscillatory flow stimulation of EOCs increased in vitro coagulation markers and ex vivo platelet accumulation. Steady flow preconditioning did not affect platelet accumulation or intimal hyperplasia relative to static samples. To determine whether in vitro markers predict implant performance, a linear regression model of the in vitro data was fit to platelet accumulation data—correlating the markers with the thromboprotective performance of the EOCs. The model was tested against implant intimal hyperplasia data and found to correlate strongly with the parallel in vitro analyses. This research defines the effects of flow preconditioning on EOC regulation of coagulation in clinical vascular grafts through parallel in vitro, ex vivo, and in vivo analyses, and contributes to the translatability of in vitro tests to in vivo clinical graft performance.     

Effects of the HIV Protease Inhibitor Ritonavir on GLUT4 Knock-out Mice

HIV protease inhibitors acutely block glucose transporters (GLUTs) in vitro, and this may contribute to altered glucose homeostasis in vivo. However, several GLUT-independent mechanisms have been postulated. To determine the contribution of GLUT blockade to protease inhibitor-mediated glucose dysregulation, the effects of ritonavir were investigated in mice lacking the insulin-sensitive glucose transporter GLUT4 (G4KO). G4KO and control C57BL/6J mice were administered ritonavir or vehicle at the start of an intraperitoneal glucose tolerance test and during hyperinsulinemic-euglycemic clamps. G4KO mice exhibited elevated fasting blood glucose compared with C57BL/6J mice. Ritonavir impaired glucose tolerance in control mice but did not exacerbate glucose intolerance in G4KO mice. Similarly, ritonavir reduced peripheral insulin sensitivity in control mice but not in G4KO mice. Serum insulin levels were reduced in vivo in ritonavir-treated mice. Ritonavir reduced serum leptin levels in C57BL/6J mice but had no effect on serum adiponectin. No change in these adipokines was observed following ritonavir treatment of G4KO mice. These data confirm that a primary effect of ritonavir on peripheral glucose disposal is mediated through direct inhibition of GLUT4 activity in vivo. The ability of GLUT4 blockade to contribute to derangements in the other molecular pathways that influence insulin sensitivity remains to be determined.     

Plasmodium knowlesi: glycolytic intermediate levels of enzyme activities of infected rhesus monkey erythrocytes

In vitro glycolytic enzyme activities and in vivo glycolytic intermediate concentrations were assayed in Plasmodium knowlesi-infected rhesus monkey erythrocytes and control erythrocytes. The enzyme activities of infected erythrocytes were greater than controls indicating that P. knowlesi had its own glycolytic system and that parasite glycolysis was the source of the increased rate of glucose consumption by infected erythrocytes. The P. knowlesi glycolytic enzymes phosphofructokinase and hexokinase were less sensitive to acid inhibition than uninfected red cells.P. knowlesi-infected monkey erythrocytes and Plasmodium berghei-infected mouse erythrocytes had similar in vivo glycolytic profiles and in vitro enzyme activity increases.