SPECTROPHOTOMETRICALLY ASSAYED INHIBITORY EFFECTS OF MERCURIC COMPOUNDS ON ANABAENA FLOS-AQUAE AND ANACYSTIS NIDULANS (CYANOPHYCEAE)1

The growth-related inhibitory effects of mercuric chloride (MC), methylmercuric chloride (MMC) and phenylmercuric acetate (PMA) (each at 1, 10, 10,² 10³ ppb) were measured in Anabaena flos-aquae (Lyng.) Bréb. and Anacystis nidulans (Richt.) Drouet & Daily. Optical density changes of control cultures compared against those of experimental cultures showed that MC was the least inhibitory of the compounds. MMC. was the most inhibitory, producing statistically significant inhibition at a concentration as low as I ppb in Anabaena. PMA was more inhibitory than MC but less than MMC. Effects caused by the mercury compounds included bleaching of individual cells, cell size changes and destruction of whole cells; the degree and extent of these effects depended on the compound and its concentration in the nutrient medium. The high sensitivities of the algae tested suggested the possibility of using them as test organisms in bioassays for mercury.     

Continuous enzymatic production and adsorption of laminaribiose in packed bed reactors

Bienzymatic production of laminaribiose from sucrose and glucose was combined with adsorption on zeolite BEA to introduce a first capture and purification step. Downstream processing including washing and desorption steps was characterized and optimized on a milliliter scale in batch mode. Results were then transferred to a packed bed system for enzymatic production and adsorption where the influence of adsorbent particle diameter on purity and productivity was evaluated. Finally, a continuous enzymatic production of laminaribiose was conducted over 10 days. The subsequent downstream processing of the loaded zeolites led to purities of over 0.5 g_{Laminaribiose} g_sugar^?1 in the desorbate with a total productivity of 5.6 mg_{Laminaribiose} L_{enzyme bed}^?1 h^?1 without the use of recycles.     

A comparison of isoprene and monoterpene emission rates from the perennial bioenergy crops short‐rotation coppice willow and Miscanthus and the annual arable crops wheat and oilseed rape

Biogenic volatile organic compounds (BVOC) emissions from bioenergy crops may differ from those of conventional crops. We compared emission rates of isoprene and a number of monoterpenes from the lignocellulosic bioenergy crops short‐rotation coppice (SRC) willow and Miscanthus, with the conventional crops wheat and oilseed rape. BVOC emission rates were measured via dynamic vegetation enclosure and GC‐MS analysis approximately monthly between April 2010 and August 2012 at a location in England and from SRC willow at two locations in Scotland. The largest BVOC emission rates were measured from willow in England and varied between years. Isoprene emission rates varied between μg g^?1 h^?1. Of the monoterpenes detected from willow, α‐pinene emission rates were highest (μg g^?1 h^?1), followed by μg g^?1 h^?1 for δ‐3‐carene, μg g^?1 h^?1 for β‐pinene and μg g^?1 h^?1 for limonene. BVOC emission rates measured in Scotland were much lower. Low emission rates of isoprene and α‐pinene were measured from Miscanthus in 2010 (μg g^?1 h^?1 and μg g^?1 h^?1, respectively) but were not detected in subsequent years. Emission rates from wheat of isoprene were negligible but relatively high for monoterpenes (μg g^?1 h^?1 and μg g^?1 h^?1 for α‐pinene and limonene, respectively). No significant emission rates of BVOCs were measured from oilseed rape. The measured emission rates followed a clear seasonal trend. Crude extrapolations based solely on data gathered here indicate that isoprene emissions from willow could correspond to 0.004–0.03% (UK) and 0.76–5.5% (Europe) of current global isoprene if 50% of all land potentially available for bioenergy crops is planted with willow.     

Effect of controlled oxygen limitation on Candida shehatae physiology for ethanol production from xylose and glucose

Carbon distribution and kinetics of Candida shehatae were studied in fed-batch fermentation with xylose or glucose (separately) as the carbon source in mineral medium. The fermentations were carried out in two phases, an aerobic phase dedicated to growth followed by an oxygen limitation phase dedicated to ethanol production. Oxygen limitation was quantified with an average specific oxygen uptake rate (OUR) varying between 0.30 and 2.48 mmolO₂ g dry cell weight (DCW)^?1 h^?1, the maximum value before the aerobic shift. The relations among respiration, growth, ethanol production and polyol production were investigated. It appeared that ethanol was produced to provide energy, and polyols (arabitol, ribitol, glycerol and xylitol) were produced to reoxidize NADH from assimilatory reactions and from the co-factor imbalance of the two-first enzymatic steps of xylose uptake. Hence, to manage carbon flux to ethanol production, oxygen limitation was a major controlled parameter; an oxygen limitation corresponding to an average specific OUR of 1.19 mmolO₂ g DCW^?1 h^?1 allowed maximization of the ethanol yield over xylose (0.327 g g^?1), the average productivity (2.2 g l^?1 h^?1) and the ethanol final titer (48.81 g l^?1). For glucose fermentation, the ethanol yield over glucose was the highest (0.411 g g^?1) when the specific OUR was low, corresponding to an average specific OUR of 0.30 mmolO₂ g DCW^?1 h^?1, whereas the average ethanol productivity and ethanol final titer reached the maximum values of 1.81 g l^?1 h^?1 and 54.19 g l^?1 when the specific OUR was the highest.     

Brewers’ spent grain liquor as a feedstock for lactate production with Lactobacillus delbrueckii subsp. lactis

Brewers’ spent grain (BSG) is a low‐cost by‐product of the brewing process. BSG liquor names the liquid components of BSG, mainly glucose, maltose, and long‐chain α‐1,4‐glycosidic bond glucose oligomers. These substances should be separated in existing BSG biorefineries, as they might lead to an increased formation of microbe‐inhibiting compounds in well‐established hydrothermal/enzymatic saccharification processes. In most cases, this liquid fraction is discarded. The present study presents for the first time an optimized process with BSG liquor for the purpose of producing bulk chemicals (e.g., lactate) in relevant concentrations. The process comprises the application of yeast extract, produced from own brewing processes, as the sole supplemented complex constituent in a simultaneous fermentation and saccharification process. Kinetic parameters for the final optimized process conditions with the organism Lactobacillus delbrueckii subsp. lactis were: maximum specific growth rate µ_max  =  0.47 h^?1, maximum lactate concentration c_Lac, max  =  79.06 g L^?1, process yield Y_PS  =  0.89 g_Lac g_Sugar^?1, lactate production rate q_P  =  4.18 g_Lac g_CDW^?1 h^?1, and productivity P _15 h  =  4.93 g_Lac L^?1 h^?1. BSG liquor, linked with yeast extract from Brewers’ yeast, can be a promising substrate for further bioprocess engineering tasks and contribute to a holistic and sustainable usage of Brewers’ spent grain.     

Influence of dietary selenite on the binding characteristics of rat serum proteins to mercurial compounds

The effect of dietary selenite on the binding characteristics of serum proteins was investigated with rats. In the control serum, the maximal binding of phenylmercuric acetate (PMA) and methylmercuric chloride (MMC) to rat serum protein was approx. 18 and 9 nmoles per mg protein, respectively. The binding of Hg²⁺ was biphasic and it did not reach a maximum at the concentrations used.Selenite treatment caused a reduction in binding capacity of serum proteins to Hg²⁺ and PMA, and an increase in the binding affinities. However, there were no such changes for the binding of MMC. Selenite protection from mercury toxicity, therefore, acts not only via a change in tissue distribution and a change in the formation of seleno-proteins but, also, via a change in the binding characteristics to some mercury compounds. In the case of methylmercury, a different mechanism of protection must exist as the modification of tissue distribution, its binding to subcellular and soluble proteins and the binding characteristics remained equivocal.     

Seasonal Growth and Photosynthetic Performance of the Antarctic Macroalga Desmarestia menziesii (Phaeophyceae) Cultivated under Fluctuating Antarctic Daylengths

Growth, photosynthesis, dark respiration and pigment contents were monitored in adult sporophytes of the Antarctic brown alga Desmarestia menziesii J. Agardh grown under fluctuating Antarctic daylength conditions. Growth rates were closely coupled to daylength variations with values varying from 0.05% d^?1 in winter condition (July-August) to 0.5% d^?1 in early summer (December). Photosynthetic pigments had maximum values of 1.8 mg g^?1 FW (chlorophyll a), 0.4 mg g^?1 FW (chlorophyll c) and 0.9 mg g^?1 FW (fucoxanthin) in summer. These changes were also closely related to individual size and biomass of the plants. Net photosynthesis (P_max), on a fresh weight basis, showed a clear seasonal pattern with highest rates of 25μmol O₂ g^?1 FW h^?1 in October and minima close to 9μmol O₂ g^?1 FW h^?1 in April. Dark respiration was high in spring (13μmol O₂ g^?1 FW h^?1) approximately coinciding with growth peaks. Likewise, photosynthetic efficiency (α) and the initial saturating light point of photosynthesis (l_k) increased significantly in spring [1.3 μimol O₂ g^?1 FW h^?1 (μmol m^?2 s^?1)^?1 and 26μmol photons m^?2 s^?1, respectively]. In the case of α, no significant differences between fresh weight and Chl a based rates were found. The results of the present study are the first that demonstrate seasonality of physiological parameters in D. menziesii sporophytes and confirm also that phenology and physiology of macroalgae can be simulated in the laboratory. On the other hand this study adds new elements to the explanation of the life strategy of D. menziesii, in particular that algal growth and photosynthesis occur under a programmed seasonal pattern.     

Uptake of glycine and release of primary amines by the polychaete Nereis virens (Sars) and the mud snail Hydrobia neglecta muus

Uptake of glycine and release of primary amines by the polychaete Nereis virens (Sars) and the mud snail Hydrobia neglecta Muus was investigated. In a flow system, Nereis virens had a net influx of glycine of 32 to 39 nmol·g^?1 fresh wt·h from a 5-μM solution of glycine. Simultaneously, an efflux of primary amines of 14 nmol·g^?1·h^?1 was measured. The experiments indicated that glycine uptake and release of primary amines are two independent processes. Glycine was absorved by Hydrobia neglecta from 50-μM solutions at an influx of 175 nmol·g^?1 organic wt·h^?1, and simultaneously, there was an independent efflux of 25 nmol primary amine·g^?1·h^?1. In the experiments, released primary amines from both species increased the ambient concentration of primary amines, but after various periods of time these concentrations apparently stabilized. Finally, the significance of micro-organisms in amino-acid uptake experiments is considered.     

Increased myocardial pyrimidine nucleotide synthesis in isoproterenol-induced cardiac hypertrophy in rats

In a first phase (up to 12^h) after the first injection of isoproterenol (5mg.kg^?1 b.w.) the pyrimidine nucleotide pools were increased and the rates of incorporation of inorganic phosphate into the α-phosphate groups of nucleotides were raised from 16 to 58 nmol.g^?1.h^?1 for uracil nucleotides and from 11 to 32 nmol.g^?1.h^?1 for cytosine nucleotides. At a later stage, while the pool sizes decreased slowly toward control levels, these rates of labelling also decreased though still remaining above control values. A similar pattern of changes was induced by the eighth daily isoproterenol injection, but the alterations were attenuated.     

Pharmacokinetics of oxytetracycline in yellow catfish [Pelteobagrus fulvidraco (Richardson, 1846)] with a single and multiple‐dose oral administration

A pharmacokinetic study of oxytetracycline (OTC) following a single (100 mg kg^?1) or a multi‐dose (100 mg kg^?1 for 5 days) oral administration was carried out in yellow catfish, Pelteobagrus fulvidraco. After oral administration at 25°C, a one‐compartment model was developed. The absorption half‐life (t_1/2(ka)) was 3.92, 1.44, 2.75, and 3.34 h in plasma, muscle, liver, and kidney after the single dose, and 0.35, 0.22, 0.42, 0.32 h after the multi‐dose, respectively. The order of peak concentration (C_max) was liver > kidney > plasma > muscle, at 3.48 μg g^?1, 2.90 μg g^?1, 1.46 μg ml^?1, and 1.39 μg g^?1 after the single dose, and 14.02 μg g^?1, 8.51 μg g^?1, 4.17 μg ml^?1, and 3.84 μg g^?1 after the multi‐dose, respectively. The elimination half‐lives (t_1/2(ke)) of OTC in plasma, muscle, liver, and kidney were calculated to be 7.64, 26.29, 19.08, and 10.61 h after the single dose, and 47.54, 70.99, 49.87, and 47.73 h after the multi‐dose, respectively. The results suggest that OTC was absorbed faster after the multi‐dose than after the single dose, suggesting that OTC could be more efficacious after the multi‐dose and more effective in the control bacterial diseases in aquaculture, with the side effects of longer withdrawal periods.     

Studies on nitrate reductase activity in Laminariadigitata (Huds.) Lamour. I. Longitudinal and transverse profiles of nitrate reductase activity within the thallus

The distribution of the enzyme nitrate reductase (NR) within the thallus of the brown alga Laminaria digitata (Huds.) Lamour is described for plants sampled from the east coast of Scotland in May and June when growth rates are at a maximum. Highest NR activities (≈ 0.2 μmol NO₃^? reduced·g^?1 wet wt·h^?1) occurred in the mature blade. NR activities declined towards the basal meristematic region of the blade. Activities in the stipe and holdfast were also low, being between 0.05 and 0.07 μmol NO₃^? reduced·g^?1 wet wt·h^?1. The activities of the enzyme glutamine synthetase (GS), which is important in the assimilation of NH₄⁺, showed a similar distribution within the blade to those of NR.The transverse profile of NR activity in the stipe exhibited a decline from the outer to the inner tissues. Maximum activities (0.13 μmol NO₃^? reduced·g^?1 wet wt·h^?1) occurred in the meristoderm, while those of the cortex and medulla were 0.04 and 0.01 μmol NO₃^? reduced·g^?1 wet wt·h^?1 respectively.These data indicate that most of the NO₃^? assimilation occurs in the mature blade rather than in the meristematic tissue where there is a high nitrogen demand for growth. The data are consistent with the maintenance of meristematic growth by the internal transport of organic nitrogen from the mature blade.     

Accumulation of phytoalexins and isoflavone glucosides in a resistant and a susceptible cultivar of Cicer arietinum during infection with Ascochyta rabiei

After infection with spores of a virulent strain of Ascochyta rabiei the chickpea (Cicer arietinum) cultivars ILC 1929 (susceptible) and ILC 3279 (resistant) were compared with regard to pterocarpan phytoalexin and isoflavone accumulation. Quantitative HPLC analyses of total extracts of aerial parts were used to measure the induced formation of the phytoalexins medicarpin and maackiain and the accumulation of the constitutive isoflavones biochanin A and formononetin together with their, 7-0-glucosides and their 7-0-glucoside-6″-0-malonates. The two cultivars showed no significant difference in the level of isoflavones and isoflavone conjugates. On the other hand, the resistant cultivar ILC 3279 rapidly accumulated large amounts of both, phytoalexins (20–26 nmole g^?1 fr.w.) whereas cultivar ILC 1929 only produced very small amounts (5 nmole g^?1 fr.w.) of medicarpin. The data are discussed with regard to isoflavonoid metabolism and the significance of induced and constitutive levels of phytoalexins and isoflavones in resistance of chickpea towards A. rabiei.     

Comparison of Methanotrophic Community and Methane Oxidation between Rhizospheric and Non-Rhizospheric Soils

Using particulate methane monooxygenase (pMMO) encoding gene, pmoA-based terminal-restrict fragment length polymorphism (T-RFLP), the methanotrophic communities between rhizospheric soils (RSs) and non-rhizospheric soil (NRSs) of landfill cover (LC), riparian wetland (RW) and rice paddy (RP) were compared before and after pre-incubation of 90 days. The ultimate potential of methane oxidation rate (UPMOR) and gene copy number of pmoA were evaluated in the soil samples after pre-incubation. Compared to the methanotrophic community in the soil samples before pre-incubation, type II methanotrophs, the Methylocystis-Methylosinus group, was mostly increased after pre-incubation, regardless of the soil type. The UPMOR (11.82 ± 0.27 μmol-CH₄· g^?1 _soil-DW·h^?1) in the LC-RS was significantly higher than that (9.57 ± 0.14 μmol-CH₄· g^?1 _soil-DW·h^?1) in the LC-NRS. However, no significant difference was found between RSs and NRSs in the RW (15.28 ± 0.91 and 13.23 ± 0.69 μmol-CH₄· g^?1 _soil-DW·h^?1, respectively) and RP (13.81 ± 1.04 and 12.81 ± 2.40 μmol-CH₄· g^?1 _soil-DW·h^?1, respectively) soils. There was no significantly difference in the gene copy numbers of pmoA in the RSs compared with those in the NRSs at all of the sampling sites. This study provides basic metagenomic information about both rhizospheric and non-rhizospheric methanotrophs, which will be helpful in developing a better strategy of biological methane removal from both natural and anthropogenic major methane sources.     

Mixed function oxidation of hexobarbital and generation of NADPH by the hexose monophosphate shunt in isolated rat liver cells.

Mixed function oxidation of hexobarbital and the generation of NADPH by the hexose monophosphate shunt were studied in isolated rat liver parenchymal cells from phenobarbital-pretreated and untreated animals. In cells isolated from untreated rats, a maximal rate of hexobarbital oxidation of 17 μmol·g^?1 liver wet weight·(60 min)^?1 was observed, while in cells isolated from phenobarbital-pretreated rats a maximal rate of 29 μmol·g^?1 liver wet weight·(60 min)^?1 has been obtained. On the basis of the specific radioactivity at carbon atom 1 of glucose 6-phosphate, fructose 6-phosphate and 6-phosphogluconate, determined by enzymatic decarboxylation, a ratio between NADPH formation via the hexose monophosphate shunt and NADH utilization for hexobarbital oxidation of 6:1 in untreated and 9.5:1 in pretreated cells has been obtained. With phenazine methosulfate the stimulation of NADPH generation via the hexose monophosphate shunt exceeded that observed in the presence of hexobarbital by 329 and 160%, respectively, indicating that the capacity of this pathway is sufficient to provide more reducing equivalents than are required for maximal rates of mixed function oxidation.     

Batch and fed-batch fermentation of optically pure D (-) lactic acid from Kodo millet (Paspalum scrobiculatum) bran residue hydrolysate: growth and inhibition kinetic modeling

Abstract

A low-cost Kodo millet bran residue was utilized as feedstock for the production of D (?) lactic acid (DLA) using Lactobacillus delbrueckii NBRC3202 under anaerobic condition. Data culled from a series of batch fermentation processes with different initial Kodo millet bran residue hydrolysate (KMBRH) and DLA concentrations were used for kinetic model development. Both simulated and experimental data were in good agreement for cell growth, KMBRH utilization, and DLA formation. The values of kinetic constants specific growth rate, (μ_m = 0.17?h^?1); growth (α_P = 0.96?g.g^?1) and non-growth (β_P = 1.19?g.g^?1.h^?1) associated constant for DLA production and the maximum specific KMBRH utilization rate, (q_{G, max} = 1.18?g.g^?1.h^?1) were in good agreement with the literature reports. Kinetic analysis elucidated that L. delbrueckii growth was predominantly influenced by KMBRH limitation and highly sensitive to DLA inhibition. Fed-batch fermentation studies demonstrated the existence of substrate and product inhibition paving the scope for process intensification.     

Methanol vapor biofiltration coupled with continuous production of recombinant endochitinase Ech42 by Pichia Pastoris

Methanol biofiltration using methylotrophic microorganisms has been previously reported by various authors. In a previous study, a modified strain of Pichia pastoris was tested for the ability to produce endochitinase (Ech42) when coupled with methanol vapor biodegradation in batch tests. The next challenge was to validate the process in a continuous system. Thus, in the present study, a biofilter packed with perlite and inoculated with P. pastoris transformed with the plasmid pPIC-ech42 was used for methanol vapor biofiltration and the continuous production of recombinant endochitinase (Ech42) for 60 days. The maximum elimination capacity (EC) of methanol obtained was 1320 g m^?3 h^?1 at a loading rate of 1465 g m^?3 h^?1. The extracellular protein production rate in the leachate was 2360 μg h^?1 with a chitinase enzymatic activity of 123 U L^?1. The protein content on the biofilm samples was negligible, indicating the effectiveness of the overall process and of P. pastoris to excrete proteins. The carbon balance indicated that 81% of the consumed methanol was mineralized and 5.8% was incorporated into biomass. The results of this study and the economic balance underscore the promising application of linking methanol vapor biofiltration to the continuous production of recombinant proteins.     

A continuous single organic phase process for the lipase catalyzed synthesis of peroxy acids increases productivity

The design of an optimal process is particularly crucial when the reactants deactivate the biocatalyst. The reaction cascades of the chemo‐enzymatic epoxidation where the intermediate peroxy acid is produced by an enzyme are still limited by enzyme inhibition and deactivation by hydrogen peroxide. To avoid additional effects caused by interfaces (aq/org) and to reduce the process limiting deactivation by the substrate hydrogen peroxide, a single‐phase concept was applied in a fed‐batch and a continuous process (stirred tank), without the commonly applied addition of a carrier solvent. The synthesis of peroxyoctanoic acid catalyzed by Candida antarctica lipase B was chosen as the model reaction. Here, the feasibility of this biocatalytic reaction in a single‐phase system was shown for the first time. The work shows the economic superiority of the continuous process compared to the fed‐batch process. Employing the fed‐batch process reaction rates up to 36 mmol h^?1 per gram_cat, and a maximum yield of 96 % was achieved, but activity dropped quickly. In contrast, continuous operation can maintain long‐term enzyme activity. For the first time, the continuous enzymatic reaction could be performed for 55 h without any loss of activity and with a space‐time yield of 154 mmol L^?1 h^?1, which is three times higher than in the fed‐batch process. The higher catalytic productivity compared to the fed‐batch process (34 vs. 18 g_Prod g^?1_cat) shows the increased enzyme stability in the continuous process.     

Activity and metabolic regulation of methane production in deep peat profiles of boreal bogs

The potential activity of methane production was determined in the vertical profiles of the peat deposits of three bogs in Tver oblast, which were representative of the boreal zone. In the minerotrophic fen, the rates of methane production measured throughout the profile did not change significantly with depth and comprised 3–6 ng CH₄-C g^?1 h^?1. In ombrotrophic peat bogs, the rate did not exceed 5 ng CH₄-C g^?1 h^?1 in the upper layer of the profile (up to 1.5 m) and increased to 15–30 ng CH₄-C g^?1 h^?1 in the deep layers of the peat deposits. The distribution of fermentative microorganisms and methanogens in the profiles of peat deposits was uniform in all the studied bogs. In bog water samples, the presence of butyrate (up to 14.1 mg 1^?1) and acetate (up to 2.4 mg 1^?1) was revealed throughout the whole profile; in the upper 0.5-m layer of the ombrotrophic bogs, formate (up to 8.9 mg 1^?1) and propionate (up to 0.3 mg 1^?1) were detected as well. The arrangement of local maxima of the fatty acid content and methanogenic activity in the peat deposits, as well as the decrease in the acetate concentrations during summer, support the hypothesis that the initial substrates for methanogenesis come from the upper peat layers. It was established that the addition of sulfate and nitrate inhibits methane production in peat samples; the changes in the concentrations, recorded in situ, may also influence the methane content in peat layers.     

Restricted permeability of rat liver for glutamate and succinate

1. When rat liver slices were incubated aerobically with [U-¹⁴C]glutamate the concentration of ¹⁴C within the slices remained lower (about 50%) than in the medium. The maximal concentration of ¹⁴C in the liver was reached within minutes. In rat kidney-cortex slices by contrast, ¹⁴C reached concentrations more than six times those of the medium. 2. In both liver and kidney ¹⁴C appeared in the respiratory CO₂, indicating penetration of glutamate carbon into the mitochondria. In kidney slices the rate of glutamate oxidation per unit weight was about five times that in liver slices. 3. Taking into account the conversion of glutamate into glucose that occurs in the kidney but not in the liver, the flux rates of glutamate through the kidney were calculated to be about 15 times those through the liver when the external glutamate concentration was 5mm. 4. Anaerobically the glutamate concentrations in medium and tissue rapidly became equal in both liver and kidney. Thus the maintenance of concentration gradients depended on the expenditure of energy. 5. [U-¹⁴C]Succinate behaved similarly to glutamate. [U-¹⁴C]Serine was taken up more rapidly by the kidney than by the liver slices, but the concentrations reached in the liver did not remain below those of the medium. [¹⁴C]Urea was distributed evenly between medium and tissue water. 6. Incubation of liver slices with [³H]inulin indicated an extracellular space of liver slices of 26%. 7. When glutamate was generated within liver slices or the perfused liver on addition of oxaloacetate, pyruvate and a source of nitrogen, the concentration of glutamate in the tissue after 1hr. was 70–97 times that in the medium. Thus the exit of glutamate from the liver cell, like its entry, is restricted. This is borne out by measurements of the specific activity of extra- and intra-cellular glutamate on addition of [U-¹⁴C]glutamate medium. 8. Liver homogenates removed added glutamate and dicarboxylic acids 20–30 times as fast as did the perfused liver. 9. It is concluded that a major permeability barrier restricts the entry and exit through the outer liver cell membrane.