Efficacy of the dietary histone deacetylase inhibitor butyrate alone or in combination with vitamin A against proliferation of MCF-7 human breast cancer cells

The combined treatment with histone deacetylase inhibitors (HDACi) and retinoids has been suggested as a potential epigenetic strategy for the control of cancer. In the present study, we investigated the effects of treatment with butyrate, a dietary HDACi, combined with vitamin A on MCF-7 human breast cancer cells. Cell proliferation was evaluated by the crystal violet staining method. MCF-7 cells were plated at 5 x 10(4) cells/mL and treated with butyrate (1 mM) alone or combined with vitamin A (10 µM) for 24 to 120 h. Cell proliferation inhibition was 34, 10 and 46% following treatment with butyrate, vitamin A and their combination, respectively, suggesting that vitamin A potentiated the inhibitory activities of butyrate. Furthermore, exposure to this short-chain fatty acid increased the level of histone H3K9 acetylation by 9.5-fold (Western blot), but not of H4K16, and increased the expression levels of p21WAF1 by 2.7-fold (Western blot) and of RARβ by 2.0-fold (quantitative real-time PCR). Our data show that RARβ may represent a molecular target for butyrate in breast cancer cells. Due to its effectiveness as a dietary HDACi, butyrate should be considered for use in combinatorial strategies with more active retinoids, especially in breast cancers in which RARβ is epigenetically altered.

Critical levels of organic acids on seed germination and seedling growth of wheat

The objective of this study was to determine the responses of the wheat cultivars CD 108 and CD 111 for tolerance to organic acids. The effects of five concentrations of the three main acids formed in the soil were studied: acetic acid (0, 4, 8, 12 and 16 mM), propionic acid (0, 4, 8, 12 and 16 mM) and butyric acid (0, 2, 4, 8 and 12 mM). Tests included germination, shoot length, root length and dry weight of shoot and root. The variable root length is the most responsive variable for all the acids tested and the critical level of toxicity of acetic, propionic and butyric acids, which reduced root length by at least 50% was 9.0, 8.5 and 4.0 mM respectively. It was concluded that the presence of acetic, propionic and butyric acids in the germination substratum of wheat seeds of the cultivars CD 111 and CD 108 reduced seedling development, mainly by reducing the length of the radicles.

Electrogenic proton/L-glutamate symport in isolated asparagus sprengeri mesophyll cell

Medium' alkaliniiation occurred -lipon the addition of L-Glu to mechanically isolated Asparagus sprenger-i mesophyll cells suspended in 1 mM CaS04. Alkalinization resulted from the coupled entry of H+ and L-Glu anion into the cells. This H+ IL-Glu symport did not stimulate K+ efflux. K+ efflux has been observed during H~ lamino acid symport in other systems. The stimulation of K+ efflux by proton coupled symport is regarded as an indicator of a plasma membrane depolarizing electrogenic symport process. H+ IL-Glu symport in Asparagus sprengerimesophyl1 cells was investigated to determine whether or not the process was electrogenic. The rate of uptake of 0.25 11M 3H-MTPP+ ( Methyltriphenylphosphonium, methyl-3H ) is a probe for monitoring changes in the membrane potential. 3HMTPP+ uptake was reduced by K+ or CCCP, agents known to depolarize the membrane potential. Uptake of 3H-MTPP+ was also inhibited by L-Glu but not by D-Glu. Conversely, 10 mM external MTPP+ inhibited the uptake of 14C-U-LGlu. Simultaneous measurements of the rates of 14C-U-L-Glu uptake and L-Glu dependent H+ influx showed that the molar stoichiometry of H+ IL-Glu symport was 2 to 1. K+ or Na+ stimulated H+ efflux was completely inhibited by DCCD, DES, oligomycin and antimycin reagents which inhibit ATP driven H+ efflux. The H+ efflux \Vas also stimulate.d by the weak acids, butyric acid and acetic acid, which are known fo-aCidify the cytoplasm. This weak acid stimulated H+ efflux was also completely inhibited by oligomycin. It was calculated that net L-Glu dependent H+ influx increased by 100% in the presence of oligomycin and that despite net medium alkalinization H+ IL-Glu symport stimulates ATP dependent H+ efflux. 11 The data presented in this study indicate that H+ IL-Glu symport is electrogenic. The data also show that ATP dependent Ht efflux rather than K+ efflux is the- process compensating for thi~ electrogenic H+ IL-Glu symport.

GABA accumulation and the hypersensitive response in isolated mesophyll cells treated with the G protein activator mastoparan

GABA (y-amino butyric acid) is a non-protein amino acid synthesized through the a-decarboxylation of L-glutamate. This reaction is catalyzed by L-glutamate decarboxylase (EC 4.1.1.15), a cytosolic Ca2+/calmodulin-stimulated enzyme. The purpose of this study is to determine whether or not GABA accumulation is associated with the hypersensitive response of isolated Asparagus sprengeri mesophyll cells. The addition of 25 J.lM mastoparan, a G protein activator, to suspensions of isolated asparagus mesophyll cells significantly increased GABA synthesis and cell death. Cell death was assessed using Evan's blue dye and fluorescein diacetate tests for cell viability. In addition, mastoparan stimulated pH-dependent alkalinization of the external medium, and a rapid and large 02 consumption followed by a loss of photosynthetic activity. The rate of 02 consumption and the net decrease in 02 in the dark was enhanced by light. The inactive mastoparan analogue Mas17 was ineffective in stimulating GABA accumulation, medium alkalinization, 02 uptake and cell death. Accumulation of H202 in response tomastoparan was not detected, however, mastoparan caused the cell-dependent degradation of added H202. The pH dependence of mastoparan-stimulated alkalinization suggests cellular electrolyte leakage, while the consumption of 02 corresponds to the oxidative burst in which 02 at the cell surface is reduced to form various active oxygen species. The results are indicative of the "hypersensitive response" of plants to pathogen attack, namely, the death of cells in the locality of pathogen invasion. The data are compatible with a model in which mastoparan triggers G protein activity, subsequent intracellular signal transduction pathway/s, and the hypersensitive response. It is postulated that the physiological elicitation of the hypersensitive response involves G protein signal transduction. The synthesis of GABA during the hypersensitive response has not been documented previously; however the role/s of GABA synthesis in the hypersensitive response, if any, remain unclear.

The production of 4-adminobutyrate in response to treatments reducing cytosolic pH and the regulation of intracellular pH

GABA (4-aminobutyrate) is synthesized through the decarboxylation of LGlu- (L-Glu-+ H+ ---> GABA + C02), and compared to many free amino acids is present in high concentrations in plant cells. GABA levels rise rapidly and dramatically in response to varied stress conditions including anaerobiosis. Recent papers suggest that GABA production and associated H+ consumption are parts of a metabolic pH-stat mechanism which ameliorates the intracellular pH decline associated with anaerobiosis or other treatments. To test this hypothesis GABA production and efflux have been measured in isolated Asparagus sprengeri cells in response to three treatments which potentially cause intracellular acidification. Acid loads were imposed using 60 min of (i) anaerobiosis, (ii) H+/LGlu- cotransport, and (iii) treatment with permeant weak acids (butyric, acetic and propionic). Both intra- and extracellular GABA concentrations increased more than 100% after anaerobiosis, almost 1000% after H+/L-Glu- cotransport (light or dark) and almost 5000/0 after addition of 5 mM butyric acid at pH 5.0. HPLC analysis of amino acids indicates that as GABA concentrations increased in response to butyric acid addition, glutamate concentrations decreased. Time-course studies demonstrated that added butyric acid stimulates GABA production by 2800/0 within 15 seconds. A fluorescent determination of cytosolic pH indicates that addition of butyric or other weak acids resulted in a rapid reduction in cytosolic pH of 0.6 pH units. The half time for the response to butyric acid addition is 2.1 seconds, indicating that the decline in cytosolic pH is rapid enough to account for the rapid stimulation of GABA production. The acid load in response to butyric acid addition was assayed by measurements of 14C-butyric acid uptake. Calculations indicate that GABA production accounted for 45% of the imposed acid load. The biological significance of GABA efflux is not yet understood. The results support the original hypothesis suggesting a role for GABA production in cellular pH regulation.

Synthèse et analyse conformationelle de dipeptides contenant l’isostère hydroxyéthylène

Dans ce mémoire, je présente mes études sur une stratégie efficace développée pour la synthèse de cétones homoallyliques substituées à partir de l’addition en cascade de réactifs de Grignard vinyliques substitués sur des α-amino esters catalysée par des sels de cuivre. L’utilisation de ces cétones homoallyliques a permis d’obtenir des mimes peptidiques comprenant un isostère de type hydroxyéthylène du lien amide. L’étape clé de cette stratégie repose sur la synthèse de cétones homoallyliques substituées intermédiaires à partir de la réaction d’additions en cascade catalysée au cuivre, de bromure de β,β-diméthylevinyle magnésium sur des analogues d’esters de la phénylalanine et de la sérine. Les cétones homoallyliques résultantes sont réduites sélectivement en alcool, la liaison double est clivée oxydativement et l’acide carboxylique résultant est couplé à un acide aminé. Afin d’évaluer l’effet qu’ont le remplacement du lien amide central dans un coude β par un hydroxyéthylène et de la présence d’un gem diméthyle sur la chaîne carbonée sur la conformation tridimensionnelle adoptée par les tripeptides générés, des analyses à l’état solide par diffraction aux rayons X, des analyses en solution par la spectroscopie RMN et des expériences de type NOESY ont été réalisées. Ces études ont permis de définir un nouveau type de coude β. La présence de pont hydrogène intramoléculaire et l’effet de restriction de conformation induit par le gem diméthyle, généralement appelé effet Thorpe-Ingold, favorisent la formation d’un coude β.

La texture et la granulométrie de la moulée influencent les concentrations d’acide propionique et butyrique de même que la prévalence d’Escherichia coli dans le tractus gastro-intestinal du porc d’engraissement

Certaines stratégies alimentaires sont actuellement considérées pour remplacer l’usage des antimicrobiens dans les fermes porcines. Les objectifs de cette étude étaient d'évaluer l'effet de la granulométrie et de la texture des aliments sur les concentrations d'acides gras volatils intestinaux, la composition des populations pathogènes et commensales d’E. coli et sur les performances de croissance des porcs. Des porcs d'engraissement (n= 840) ont reçu l'une des six diètes suivantes: moulée texturée 500, 750 et 1250 µm et moulée cubée 500, 750 et 1250 µm. Le gain de poids a été mesuré à chaque changement de formulation de moulée. À l'abattoir, les contenus du caecum et du côlon de 165 porcs ont été échantillonnés pour le dénombrement des E. coli par PCR quantitatif (qPCR) et pour la quantification des AGV. Le gène yccT a été utilisé pour dénombrer les E. coli totaux. Une diminution du taux de conversion alimentaire a été associée avec la moulée cubée et/ou la moulée de 500 µm. Les concentrations d’acide propionique et butyrique, et ce tant au niveau du caecum que du côlon, étaient plus élevées chez les porcs recevant de la moulée texturée que chez ceux recevant de la moulée cubée. Du point de vue de la granulométrie, les concentrations caecales et du côlon d’acide butyrique étaient plus élevées chez les porcs alimentés avec de la moulée de 1250 µm que chez ceux recevant de la moulée de 500 µm. D'autre part, les niveaux intestinaux d’E. coli totaux étaient plus élevés pour les porcs nourris avec de la moulée cubée que pour ceux ayant reçu de la moulée texturée. Les résultats ont montré que la moulée texturée est associée à des performances de croissance plus faibles mais à des changements intestinaux favorables.

Neurotransmitter Functional Role in Neurodegenerative Diseases: human Health

Neuroscience is the study of'tbe ne rvous system , including the i - ; . in, spinal cord and peripheral nerves . Neurons are the basic cells of the brain and nervous system which exerts its functional role through various neurotransmitters and receptor systems . The activity of a nen ren depends on the balance between the number of excitatory and inhibito r y processes affecting it, both processes occurring individually and sin ,tlte-' ,ieously. The functional bal,ince of different neurotransmitters such as Acct >>lcholine (Ach), Dopamine (DA), Serotonin (5-1-17), Nor epinepbri,te (N.1 j, Epinephrine (LPI), Glutamate and Gamma amino butyric acid (GA BA) regulates the growth , division and other vital functions ofa normal cell / organisin (Sudha, 1 998). The micro-environ ; nertt of the cell is controlled / the macro-environment that surrounds the individual. Any change in the cell environment causes imbalance in cell homeostasis and f,ntction. Pollution is a significant cause of imbalance caused iii the inacYcenvironment. Interaction with polluted environments can have an adverse impact on the health of humans. The alarming rise in enviromilmieil cont.iniin :rtion has been linked to rises in levels of pesticides, ndltstr al effluents, domestic Waste, car exhausts and other anthropogenic activities. Persistent exposures to contaminant cause a negative imp,-, on brain health and development . Pollution also causes a change in the neurotransmitters and their receptor function leading to earl.;' recurrence of neurodcge,terative disorders such as flypoxia , Alzbeimers's and Huntington 's disease early in life.

Neurotransmitters Functional Balance in Neurodegenerative Disease Management:recent Advances

The recent developments in neurobiology have rendered new prominence and potential to study about the structure and function of brain and related disorders. Human behaviour is the net result of neural control of the communication between brain cells. Neurotransmitters are chemicals that are used to relay, amplify and modulate electrical signals between neurons and/or another cell. It mediates rapid intercellular communication through the nervous system by interacting with cell surface receptors. These receptors often trigger second messenger signaling pathways that regulate the activity of ion channels. The functional balance of different neurotransmitters such as Acetylcholine (Ach), Dopamine (DA), Serotonin (5-HT), Norepinephrine (NE), Epinephrine (EPI), Glutamate and Gamma amino butyric acid (GABA) regulates the growth, division and other vital functions of a normal cell / organism (Sudha, 1998). Any change in neurotransmitters' functional balance will result in the failure of cell function and may lead to the occurrence of diseases. Abnormalities in the production or functioning of neurotransmitters have been implicated in a number of neurological disorders like Schizophrenia, Alzheimer's, Epilepsy, Depression and Parkinson's disease. Changes in central and peripheral neuronal signaling system is also noted in diabetes, cancer, cell proliferation, alcoholism and aging. Elucidation of neurotransmitters receptor interaction pathways and gene expression regulation by second messengers and transcriptional factors in health and disease conditions can lead to new small molecules for development of therapeutic agents to improve neurological disease conditions. Increased awareness of the global effects of neurological disorders should help health care planners and the neurological community set appropriate priorities in research, prevention, and management of these diseases.

Evaluation of Somaclonal Variation in Callus Cultures of Jatropha curcas Maintained on Different Hormonal Combinations Using RAPD Markers

Random genetic changes generated during in vitro culture are not desirable for plant micropropagation and genetic transformation. RAPD markers were used to detect the variation in leaf disc callus cultures of Jatropha curcas, maintained in Murashige and Skoog (MS) medium with different auxin and cytokinin combinations. In total 41 scorable bands were produced with 11 primers. Out of 41 bands, 37 were polymorphic (91.12%). The average number of polymorphic bands was 3.36 per primer. The highest similarity (0.82) with mother plant was seen in callus maintained on MS with hormonal combination Indole butyric acid - 0.4mg/l+ N6-benzyladenine purine - 4.0 mg/l. The callus grown on MS with hormonal combinations IBA- 0.4mg/l+ BAP- 2.0mg/l, IBA- 0.4mg/l+ BAP- 2.5mg/l and IBA- 0.6 mg/l+ BAP- 2.0 mg/l also showed similarity with the mother plant. Callus maintained on MS with hormonal combination IBA- 0.2mg/l+ BAP- 2.0 mg/l was found to show least similarity (0.53) with mother plant

Biobutanol from lignocellulosic biomass by a novel Clostridium sporogenes BE01

In the current study, a novel non-acetone forming butanol and ethanol producer Was isolated and identified. Based on the 16s rDNA sequence BLAST and phylogenetic analyses, it was found to have high similarity with the reported hydrogen producing strains of Clostridium sporogenes. Biochemical studies revealed that it is lipase and protease positive. The lipolytic and proteolytic properties are the very important characteristics of Clostridium sporogenes. Sugar utilization profile studies were positive for glucose, saccharose, cellobiose and weakly positive result to xylose. This study demonstrated C. sporogenes BE01, an isolate from NIIST is having potential to compete with existing, well known butanol producers with the advantage of no acetone in the final solvent mixture. Rice straw hydrolysate is a potent source of substrate for butanol production by C. sporogenes BE01. Additional supplementation of vitamins and minerals were avoided by using rice straw hydrolysate as substrate. Its less growth, due to the inhibitors present in the hydrolysate and also inhibition by products resulted in less efficient conversion of sugars to butanol. Calcium carbonate played an important role in improving the butanol production, by providing the buffering action during fermentation and stimulating the electron transport mediators and redox reactions favoring butanol production. Its capability to produce acetic acid, butyric acid and hydrogen in significant quantities during butanol production adds value to the conversion process of lignocellulosic biomass to butanol. High cell density fermentation by immobilizing the cells on to ceramic particles improved the solvents and VFA production. Reduced sugar utilization from the concentrated hydrolysate could be due to accumulation of inhibitors in the hydrolysate during concentration. Two-stage fermentation was very efficient with immobilized cells and high conversions of sugars to solvents and VFAs were achieved. The information obtained from the study would be useful to develop a feasible technology for conversion of lignocellulosic biomass to biobutanol.

Etablierung neuer Methoden zur in vitro Kultivierung der Baumart Fraxinus excelsior L. und Entwicklung von Verfahren zur Kryokonservierung von in vitro Sprossspitzen

Die hier vorliegende Arbeit wurde im Rahmen eines europäischen Projektes mit dem Titel „Improving Fraxinus (Ash) productivity for European needs by testing, selection, propagation and promotion of improved genetic resources“ an der Niedersächsischen Forstlichen Versuchsanstalt, Abteilung Waldgenressourcen erstellt. Im Rahmen des Projektes wurden 62 Plusbäume aus einem 15 Jahre alten europäischen Herkunfts-/ Nachkommenschaftsversuch in den Niedersächsischen Forstämtern Bovenden und Dannenberg nach den Kriterien Stammform und Wuchsleistung für die vegetative Vermehrung ausgewählt. Ziel dieser Arbeit war die Optimierung bestehender in vitro Protokolle sowie die Entwicklung eines bisher noch nicht existierenden Kryokonservierungsprotokolls für in vitro Sprossspitzen. Im ersten Teil dieser Arbeit wird die Entwicklung des in vitro Protokolls für Fraxinus excelsior dargestellt. Die Optimierung der Methoden zur Etablierung, Vermehrung und Bewurzelung erfolgte durch Versuchsreihen mit unterschiedlichen Klonen, so dass insgesamt 26 der selektierten Plusbäume erfolgreich in vitro etabliert werden konnten. Achselknospen frischer Triebe der Pfropflinge der Mutterbäume stellten die beste Explantatquelle dar. Die Explantate wurden mit 0,2 % Quecksilberchlorid (HgCl2) oberflächensterilisiert bevor sie auf hormonfreies Woody Plant Medium (WPM) transferiert wurden. Nach zwei Wochen erfolgte ein Transfer auf WPM mit 4 mg/l 6-Benzylaminopurine (BAP) und 0,15 mg/l Indole-3-butyric acid (IBA). Die besten Vermehrungsraten wurden auf WPM mit 4 mg/l BAP, 0,15 mg/l IBA und 0,01 mg/l TDZ und 0,7 % Agar in Honiggläsern mit einem Plastikdeckel erzielt. Als Bewurzelungsmedium wurde 0,5 konzentriertes Murashige und Skoog (MS) Medium mit 2 mg/l IBA, 0,25 mg/l BAP und 0,8 % Agar verwandt. Im zweiten Teil der Arbeit werden die Versuchsreihen zur Entwicklung des Kryokonservierungsprotokolls von in vitro Sprossspitzen dargestellt. Zur Entwicklung der Methode wurden die Vorbehandlungsbedingungen verbessert und zwei Techniken, die Alginat- / Dehydrati-onsmethode und die Vitrifikationsmethode mit Hilfe der sogenannten PVS2-Lösung (Plant Vitrification solution number 2) getestet. Die optimierte PVS2-Methode erwies sich als die für Esche besser geeignete Technik und ließ sich erfolgreich zur Kryokonservierung juveniler und adulter Kulturen anwenden. Die Regenerationsraten lagen zwischen 50 und 100 % für juvenile bzw. 50 und 80 % für adulte Kulturen.

Digestion, rumen fermentation and circulating concentrations of insulin, growth hormone and IGF-1 in steers given maize silages harvested at three stages of maturity

Advancing maturity of forage maize is associated with increases in the proportion of dry matter (DM) and starch and decreases in the proportions of structural carbohydrates in the ensiled crop. Three maize silages (286 (low, L), 329 (medium, M) and 379 (high, H) g DM per kg fresh weight) plus a concentrate formulated to give isonitrogenous intakes were offered to Holstein-Friesian steers fitted with a cannula in the dorsal sac of the rumen and a 'T' piece cannula in the proximal duodenum in an experiment with a cross-over design that allowed four collection periods. Nutrient flow to the duodenum was estimated using chromium-EDTA. Steers consumed approximately 0(.)6 kg DM per day less of diet L compared with the other two diets (P=0(.)026), resulting in less DM being digested (P=0(.)005) but digestibility did not differ between diets. Similar results were obtained for organic matter. There were no differences between diets in the intake or digestibility of neutral-detergent fibre. Intake, duodenal flow and faecal output of starch were greater for steers offered diets M and H compared with those given diet L (P < 0(.)05). In all diets rumen digestion contributed to over 90% of total digestion of starch, although rumen digestibility declined significantly with advancing maize maturity (P=0(.)002). Molar proportions of acetic acid were higher in diet H (P < 0(.)05) whilst proportions of propionic acid and n-butyric acid were higher in diets M and L. There were no significant differences between diets in mean rumen pH or ammonia concentrations. Mean circulating concentrations of insulin were higher (P=0(.)009) in cattle given diets L and M compared with diet H. There were no differences between diets in the mean circulating concentration of growth hormone, or the frequency, amplitude and duration of growth hormone pulses, or the mean circulating concentrations of IGF-1. Changes in forage composition that accompany advancing maize maturity affect overall silage digestion and circulating concentrations of insulin.

Rates of production of acetate, propionate, and butyrate in the rumen of lactating dairy cows given normal and low-roughage diets

Five lactating dairy cows with a permanent cannula in the rumen were given ( kg DM/d) a normal diet (7.8 concentrates, 5.1 hay) or a low-roughage (LR) diet (11.5 concentrates, 1.2 hay) in two meals daily in a two-period crossover design. Milk fat (g/kg) was severely reduced on diet LR. To measure rates of production of individual volatile fatty acids (VFA) in the rumen, 0.5 mCi 1-C-14-acetic acid, 2-C-14-propionic acid, or 1-C-14-n-butyric acid were infused into the rumen for 22 h at intervals of 2 to 6 d; rumen samples were taken over the last 12 h. To measure rumen volume, we infused Cr-EDTA into the rumen continuously, and polyethylene glycol was injected 2 h before the morning feed. Results were very variable, so volumes measured by rumen emptying were used instead. Net production of propionic acid more than doubled on LR, but acetate and butyrate production was only numerically lower. Net production rates pooled across both diets were significantly related to concentrations for each VFA. Molar proportions of net production were only slightly higher than molar proportions of concentrations for acetate and propionate but were lower for butyrate. The net energy value (MJ/d) of production of the three VFA increased from 89.5 on normal to 109.1 on LR, equivalent to 55 and 64% of digestible energy, respectively. Fully interchanging, three-pool models of VFA C fluxes are presented. It is concluded that net production rates of VFA can be measured in non-steady states without the need to measure rumen volumes.

Root pruning as a means to encourage root growth in two ornamental shrubs, Buddleja davidii ‘Summer Beauty’ and Cistus ‘Snow Fire'

The inability of a plant to grow roots rapidly upon transplanting is one of the main factors contributing to poor establishment. In bare-rooted trees, treatments such as root pruning or application of the plant hormone auxin [e.g., indole butyric acid (IBA)] can promote root growth and aid long-term establishment. There is little information on ornamental containerised plants, however, other than the anecdotal notion that 'teasing' the roots out of the rootsoil mass before transplanting can be beneficial. In the present study we tested the ability of various root-pruning treatments and application of IBA to encourage new root and shoot growth in two shrub species, commonly produced in containers - Buddleja davidii 'Summer Beauty' and Cistus 'Snow Fire'. In a number of experiments, young plants were exposed to root manipulation (teasing, light pruning, or two types of heavy pruning) and/or treatment with IBA (at 500 or 1,000 mg l-1) before being transplanted into larger containers containing a medium of 1:1:1 (v/v/v) fine bark, sand and loam. Leaf stomatal conductance (gl) was measured 20 min, and 1, 2, 4 and 6 h after root manipulation. Net leaf CO2 assimilation (A) was measured frequently during the first week after transplanting, then at regular intervals up to 8 weeks after transplanting. Plants were harvested 8 weeks after transplanting, and root and shoot weights were measured. In both species, light root pruning alone, or in combination with 500 mg l-1 IBA, was most effective in stimulating root growth. In contrast, teasing, which is commonly used, showed no positive effect on root growth in Buddleja, and decreased new root growth in Cistus. The requirement for exogenous auxin to encourage new root growth varied between experiments and appeared to be influenced by the age and developmental stage of the plants. There were no consistent responses between root treatments and net CO2 assimilation rates, and changes in root weight were not closely correlated with changes in assimilation. The mechanisms whereby new root growth is sustained are discussed.