Effects of Cadmium on Root Growth, Cell Division and Nucleoli in Root Tip Cells of Garlic

The effects of different concentrations (10⁻⁷ to 10⁻² M) of cadmium chloride on root growth, cell division and nucleoli in root tip cells of Allium sativum L. were investigated. At lower concentrations of Cd²⁺ (10⁻⁷ to 10⁻⁶ M), Cd²⁺ did not influence the root growth, even had a stimulation effects during a short treatment. The results showed that the rate of root growth per day at the treatment groups (10⁻⁴ to 10⁻² M Cd²⁺) decreased with increasing duration of the treatment and increasing Cd²⁺ concentration. Cd²⁺ induced c-mitosis, anaphase bridges, chromosome stickiness and on nucleoli, causing some particles of similar silver-stained material scattered in the nuclei and making the silver staining reaction at the periphery of the nucleolus weaker. This revised version was published online in July 2006 with corrections to the Cover Date.     

The effect of cadmium on potato (Solanum tuberosum L.) shoot culture growth

The uptake of cadmium and its effect on the growth of potato shoot tips grownin vitro were followed in dependence on cadmium concentration in nutrient medium. Concentration of 10 ⁻⁶ M Cd ²⁺ did not substantially affect potato plantlet growth dynamics; but the concentration of 10⁻³ M Cd²⁺ showed a strong growth inhibitory effect accompanied with increased cadmium accumulation in both root and shoot tissues.     

Attenuation of cadmium toxicity in mycorrhizal celery (<Emphasis Type="Italic">Apium graveolens</Emphasis> L.)

A pot-culture experiment was carried out to investigate the effect of arbuscular mycorrhizal (AM) fungus (Glomus macrocarpum Tul. and Tul.) on plant growth and Cd²⁺uptake by Apium graveolens L. in soil with different levels of Cd²⁺. Mycorrhizal (M) and non-mycorrhizal (NM) plants were grown in soil with 0, 5, 10, 40 and 80 Cd²⁺ mg kg⁻¹soil. The infectivity of the fungus was not affected by the presence of Cd²⁺ in the soil. M plants showed better growth and less Cd²⁺ toxicity symptoms. Cd²⁺ root : shoot ratio was higher in M plants than in NM plants. These differences were more evident at highest Cd²⁺ level (80 mg kg⁻¹ soil). Chlorophyll a and chlorophyll b concentrations were significantly higher in AM-inoculated celery leaves. The dilution effect due to increased biomass, immobilization of Cd²⁺ in root and enhanced P-uptake in M plants may be related to attenuation of Cd²⁺toxicity in celery.     

Effects of metal ions on activity of plasmin

The effects of some metal ions on amidolytic and fibrinogenolytic activities of highly purified human plasmin were investigated in vitro. In the presence of Zn²⁺, Cu²⁺, Cd²⁺, and Au⁺ in the incubation mixture at the concentrations of 1×10⁻⁵−1×10⁻³ M, the anidolytic plasmin activity was strongly inhibited, whereas Ca²⁺ and Mg²⁺ at the same concentrations were not effective. The analysis of the kinetic study has shown that Zn²⁺ or Cu²⁺ acts as mixed-type inhibitors of plasmin activity. The inhibition of amidolytic plasmin activity by Zn²⁺ and Cu²⁺ was reduced in the presence of EDTA, histidine, or albumin. Incubation of plasmin with Zn²⁺ or Cu²⁺ (at the concentration of 5×10⁻⁴ M) resulted in complete loss of its proteolytic action on fibrinogen, whereas Cd²⁺ and Au⁺ under the same conditions only partially inhibited this process.     

The Activity of the Antioxidative System in Cadmium-Treated Arabidopsis thaliana

Changes in the content of reactive oxygen species (ROS) and activity of the antioxidant system were measured in leaves of Arabidopsis thaliana (L.) Heynh exposed to Cd²⁺. Mature plants growing in the nutrient solution were treated with Cd²⁺ at different concentrations (0, 5, 25, 50, 100 μM). An increase of content in leaves was observed at 5, 25 and 50 μM Cd²⁺. A strong accumulation of H₂O₂ was found only at the lowest Cd²⁺ concentration. The content of OH^*. was high at 50 and 100 μM Cd²⁺. Superoxide dismutase (SOD) activity was always higher in Cd²⁺-treated plants than in control. Catalase (CAT) activity decreased with increasing Cd²⁺ concentration in the nutrient solution. Guaiacol peroxidase (POX) activity was particularly high at the lowest and highest Cd²⁺ concentrations and ascorbate peroxidase (APX) activity additionally at 50 μM Cd²⁺. Enhanced activity of monodehydroascorbate reductase (MDHAR) and strong reduction in ascorbate (AA) content were observed at 25 μM Cd²⁺. Glutathione reductase (GR) activity was always higher than in control but decreased as Cd²⁺ concentration increased. However, it was accompanied by gradual content increase of SH-groups. This revised version was published online in July 2006 with corrections to the Cover Date.     

Salinity affects indirectly nitrate acquisition associated with glutamine accumulation in cowpea roots

The aim of this study was to test the hypothesis that salinity can affect indirectly the nitrate acquisition by a negative modulation triggered by glutamine accumulation. Cowpea plants were exposed to a mild NaCl concentration (50 mM) in order to restrict growth and N-demand. After 21 d, pretreated plants and control plants were supplied with 0, 5 and 10 mM of Ca(NO₃)₂ for 3 d in absence of NaCl. Salt pretreated plants showed a great limitation in acquisition of NO₃ ⁻, indicated by decline in the nitrate uptake rate, NO₃ ⁻ accumulation, nitrate reductase activity and protein content. The restriction of NO₃ ⁻ utilization was positively associated with increased glutamine synthetase activity and glutamine accumulation, especially in roots.     

The toxicity of cadmium to barley plants as affected by complex formation with humic acid

An ‘alternating solution’ culture method was used to study the effects of chloride ions and humic acid (HA) on the uptake of cadmium by barley plants. The plants were transferred periodically between a nutrient solution and a test solution containing one of four levels of HA (0, 190, 569 or 1710 μg cm⁻³) and one of five levels of Cd (0, 0.5, 1.0, 2.5 or 5.0 μg cm⁻³) in either a 0.006M NaNO₃ or 0.006M NaCl medium. Harvest and analysis of shoots and roots was after nineteen days. The distribution of Cd in the test solutions between Cd²⁺, CdCl⁺ and HA-Cd was determined in a separate experiment by dialysis equilibrium. In the nitrate test solutions Cd uptake was clearly controlled by Cd²⁺ concentration and was therefore reduced by HA complex formation. In the absence of HA, chloride suppressed Cd uptake indicating that Cd²⁺ was the preferred species. However complex formation with Cl⁻ enhanced uptake when HA was present because of an increase in the concentration of inorganic Cd species relative to the nitrate system. The ratio root-Cd/shoot-Cd remained at about 10 across a wide range of shoot-Cd concentrations, from about 3 μg g⁻¹ (sub-toxic) up to 85 μg g⁻¹ (80% yield reduction). The ability of the barley plants to accumulate ‘non-toxic’ Cd in their roots was thus very limited. Humic acid also had no effect on Cd translocation within the plant and the root/shoot weight ratio did not vary with any treatment. At shoot-Cd concentrations in excess of 50 μg g⁻¹, K, Ca, Cu and Zn uptake was reduced, probably the result of root damage rather than a specific ion antagonism. The highest concentration of HA also lowered Fe and Zn uptake and there was a toxic effect with increasing HA concentration at Cd=0. However the lowest HA level, comparable with concentrations found in mineral soil solutions, only reduced yield (in the absence of Cd) by <5% while lowering Cd uptake across the range of Cd concentrations by 66%–25%.     

Different characteristics of roots in the cadmium-tolerance and Cd-binding complex formation between mono- and dicotyledonous plants

Effects of Cd²⁺ on growth and Cd-binding complex formation in roots were examined with various seedlings of mono- and dicotyledonous plants. Maize, oat, barley and rice exhibited the greater tolerance to Cd²⁺ (100 μM) than did azuki bean, cucumber, lettuce, pea, radish, sesame and tomato (10–30 μM). Azuki bean was the most sensitive to Cd²⁺ (<10 μM). Under these Cd-treatments, cereal roots accumulated Cd²⁺ in the cytoplasmic fractions and transported Cd²⁺ into the same fractions of shoot tissues, to larger extents than did dicotyledonous roots. Cereal roots synthesized a Cd-binding complex containing phytochelatins in the cytoplasmic fractions, depending upon Cd²⁺ concentrations applied (30–100 μM). Such a complex was not detected from the same fractions of dicotyledonous roots treated with Cd²⁺. These results suggest that the Cd-binding complex formation has an important role in the tolerance of cereal roots against Cd²⁺.     

Cd-induced growth reduction in the halophyte <Emphasis Type="Italic">Sesuvium portulacastrum</Emphasis> is significantly improved by NaCl

The effects of Cd²⁺ and NaCl, applied together or separately, on growth and uptake of Cd²⁺ were determined for the halophyte Sesuvium portulacastrum L. Seedlings were cultivated in the presence of 50 or 100 μmol L⁻¹ Cd²⁺ alone or combined with 100 or 400 mmol L⁻¹ NaCl. Data showed that alone, Cd²⁺ induced chlorosis, necrosis, and inhibited growth. Addition of NaCl to Cd²⁺-containing medium restored growth and alleviated the toxicity, however. NaCl also enhanced the amounts of Cd²⁺ accumulated in the shoots. All Cd²⁺ treatment reduced K⁺ and Ca²⁺ uptake and transport to the shoots. Accumulation of Na⁺ in the shoots was not affected by Cd²⁺, however. Thus S. portulacastrum maintained its halophytic characteristics in the presence of Cd²⁺. We suggest this halophyte could be used for phytoextraction of Cd²⁺ from salt-contaminated sites.     

<Emphasis Type="Italic">Tolypothrix tenuis</Emphasis> stress response to nickel

Summary Metal ions are both essential and potentially toxic. The aim of this work was to demonstrate that diazotrophic cyanobacterium Tolypothrix tenuis N° 54 can tolerate toxic concentrations of Ni²⁺ in order to use the biomass in biofilters or as biofertilizer. For this purpose, growth, pigment and protein contents and catalase activity of T. tenuis growing in increasing concentrations of Ni²⁺ ranging from 10⁻¹⁰ to 10⁻⁴ M were assesed. The strain did not grow at Ni²⁺ concentration of 10⁻⁴ M, but at lower concentrations there were no significant differences with the control; it was tolerant at 10⁻¹⁰ and 10⁻⁸ M. Nickel concentration of 10⁻⁶ M is toxic for this cyanobacterial strain, because dry weight decreased by 30%; allophycocyanin and phycoerythrin decreased by 92% and 98%, respectively and protein content increased by 42%. Chlorophyll a concentration was more than double the control value in 10⁻¹⁰ and 10⁻⁸ M, but in 10⁻⁶ M it decreased by 19%. Catalase (E.C. 1.11.1.6) activity doubled the control value in the lowest nickel concentration whereas in 10⁻⁸ M there was no significant difference with the control and in 10⁻⁶, it decreased by 78%. The living biomass of this strain could be used as a step in the bioremediation process in waters contaminated with concentrations of nickel lower than 10⁻⁶ M and eventually as a biofertilizer.     

Effect of Cadmium on gamma-Glutamylcysteine Synthesis in Maize Seedlings

Cysteine, γ-glutamylcysteine, and glutathione and the extractable activity of the enzymes of glutathione biosynthesis, γ-glutamylcysteine synthetase (EC 6.3.2.2) and glutathione synthetase (EC 6.3.2.3), were measured in roots and leaves of maize seedlings (Zea mays L. cv LG 9) exposed to CdCl₂ concentrations up to 200 micromolar. At 50 micromolar Cd²⁺, γ-glutamylcysteine contents increased continuously during 4 days up to 21-fold and eightfold of the control in roots and leaves, respectively. Even at 0.5 micromolar Cd²⁺, the concentration of γ-glutamylcysteine in the roots was significantly higher than in the control. At 5 micromolar and higher Cd²⁺ concentrations, a significant increase in γ-glutamylcysteine synthetase activity was measured in the roots, whereas in the leaves this enzyme activity was enhanced only at 200 micromolar Cd²⁺. Labeling of isolated roots with [³⁵S]sulfate showed that both sulfate assimilation and glutathione synthesis were increased by Cd. The accumulation of γ-glutamylcysteine in the roots did not affect the root exudation rate of this compound. Our results indicate that maize roots are at least in part autonomous in providing the additional thiols required for phytochelatin synthesis induced by Cd.     

Kinetic Analysis of the Calcium- and Cadmium-Induced Development of Nonspecific Permeability of the Mitochondrial Inner Membrane

We studied the Са²⁺- and Cd²⁺-induced development of the nonspecific permeability of the mitochondrial inner membrane in preparations obtained from rat liver tissue, which is accompanied by swelling of these organelles and intensification of light dispersion of their suspension. Addition of 5 to 100 μM Са²⁺ or 1 to 50 μM Сd²⁺ to the medium caused swelling of the mitochondria. With increase in concentrations of Са²⁺ and Cd²⁺, the latency of the effect decreased, and the rate of swelling of these organelles increased. Upon isolated action of Са²⁺, the intensity of the process (amplitude of changes) did not depend significantly on the concentration of the above ions, while upon isolated action of Cd²⁺, it was the maximum at the concentration of 1 mM and noticeably decreased with increase in the concentration. The dependence of the rate of Са²⁺- and Cd²⁺-induced swelling of the mitochondria on the concentration of these ions was described by power and sigmoid functions, respectively. The calculated maximum rate and the constant of 50% saturation of these processes were equal to 0.609 and 1.084 extinction units/min⋅mg protein and 19.85 and 7.28 μM for Са²⁺- and Cd²⁺-induced swelling of the mitochondria, respectively. Cyclosporine A (10 μM) suppressed completely the Са²⁺-induced swelling of the mitochondria and decreased only partly the Cd²⁺-induced swelling. Dithiothreitol (1 mM) inhibited completely the latter effect but did not influence significantly the Са²⁺-stimulated process. Therefore, the distinctions between the kinetics of Са²⁺- and Cd²⁺-induced swelling of the mitochondria, as well as the different sensitivity of these processes to cyclosporine A and dithiothreitol, prove that the mechanisms underlying interactions between the cations of the above metals and the inner mitochondrial membrane in the course of the development of nonspecific permeability of these organelles are dissimilar. *Deceased     

Lhcb2 gene expression analysis in two ecotypes of Sedum alfredii subjected to Zn/Cd treatments with functional analysis of SaLhcb2 isolated from a Zn/Cd hyperaccumulator

The Lhcb2 gene from hyperaccumulator Sedum alfredii was up-regulated more than three-fold while the non-hyperaccumulator accumulated one or two-fold higher amount of the mRNA than control plants under different concentrations of Cd²⁺ for 24 h. Lhcb2 expression was up-regulated more than five-fold in a non-hyperaccumulator S. alfredii when exposed to 2 μM Cd²⁺ or 50 μM Zn²⁺ for 8 d and the hyperaccumulator had over two-fold more mRNA abundance than the control plants. Over-expression of SaLhcb2 increased the shoot biomass by 14–41% and the root biomass by 21–57% without Cd²⁺ treatment. Four transgenic tobacco lines (L5, L7, L10 and L11) possessed higher shoot biomass than WT plants with Cd²⁺. Four transgenic lines (L7, L8, L10 and L11) accumulated 6–35% higher Cd²⁺ amounts in shoots than the wild type plants.     

Differences in the binding modes of phytochelatin to cadmium (II) and Zinc(II) ions

An ¹H NMR (nuclear magnetic resonance) spectroscopic structural analysis of Cd²⁺ complexes formed with the pentapeptide phytochelatin, (NH₃)⁺−(ψ-Glu-Cys)₂−Gly−COO−(PC₂), at a pH of 7.5 showed that the two thiol groups of the Cys residues and either the carbonyl or amide group of the peptide bond between Glu1 and Cys1 act as possible donor groups in the complexes at Cd²⁺/PC₂ ratios up to 0.4. As the ratio increases, the carboxylate group of Glu2 and either the carbonyl or amide group of the peptide bond between Cys1 and Glu2 comes to serve as a donor group. The manner in which Cd²⁺ forms complexes with PC₂ is distinctly different from Zn²⁺ and might account for the role of phytochelatin in Cd²⁺ detoxification. Electron absorption spectrometry demonstrated that the Cd²⁺ complexes are coordinated in a tetrahedral fashion by four thiol groups and that several sulfur atoms might bridge Cd²⁺ ions, resulting in the formation of polynuclear complexes. This contrasts with Zn²⁺ complex formation, which consists exclusively of a 1:1 complex.     

Metal resistance and removal by two strains of the green alga, <Emphasis Type="Italic">Chlorella vulgaris</Emphasis> Beijerinck,isolated from Laguna de Bay,Philippines

Two strains of Chlorella vulgaris Beijerinck isolated from two different sites in Laguna de Bay, Philippines, were studied for their resistance and ability to remove four metal ions, i.e., Cu²⁺, Cr⁶⁺, Pb²⁺, and Cd²⁺ added separately in BG-11 growth medium. The growth of the two strains was severely inhibited at 2 mg.L⁻¹ of Cu²⁺, 5 mg.L⁻¹ of Cr⁶⁺, 8 mg.L⁻¹ of Pb²⁺, and 10 mg.L⁻¹ of Cd²⁺. However, the two strains exhibited different EC₅₀ values for the same metal ion. The WB strain had a significantly higher resistance (p < 0.01) for Cd²⁺ and Cr⁶⁺ compared with the SB strain, while the SB strain had significantly higher resistance (p < 0.01) for Cu²⁺ compared with the WB strain. On the other hand, the two strains behaved differently in their capacity to remove the metal ions in BG-11 medium containing 1.0 mg.L⁻¹ of the three metal ions, except for Cu²⁺, which was added at 0.1 mg.L⁻¹. The WB strain showed the highest removal of Cd²⁺ at 70.3% of total, followed by Pb²⁺ at 32%, while the SB strain exhibited the highest removal of Pb²⁺ at 48.7% followed by Cd²⁺ at 40.7% of the total. Both strains showed the least removal of Cr⁶⁺ at 28% and 20.8% of the total for the WB and SB strains respectively. The percentage removal for Cu²⁺ was 50.7% and 60.8% for the WB and SB strains respectively. After 12 days of incubation, both strains showed that a greater percentage of the metal ions removed were accumulated intracellularly than adsorbed at a ratio of at least 2:1. Both strains manifested the same cytological deformities, like a loss of pyrenoids at 10 mg.L⁻¹ in all four metal ions. Discoloration and disintegration of chloroplasts were observed at 1.0 mg.L⁻¹ in Cu²⁺ and 5 mg.L⁻¹ in Cr⁶⁺. The nonrelease of autospores from the mother cells was observed at 10 mg.L⁻¹ in Cu²⁺ and Cr⁶⁺. Presented at the 6th Meeting of the Asian Pacific Society of Applied Phycology, Manila, Philippines.     

Resveratrol Increases Glutamate Uptake, Glutathione Content, and S100B Secretion in Cortical Astrocyte Cultures

Resveratrol (3,5,4′-trihydroxy-trans-stilbene) is a polyphenol present in grapes and red wine, which has antioxidant properties and a wide range of other biological effects. In this study, we investigated the effect of resveratrol, in a concentration range of 10–250 μM, on primary cortical astrocytes; evaluating cell morphology, parameters of glutamate metabolism such as glutamate uptake, glutamine synthetase activity and glutathione total content, and S100B secretion. Astrocyte cultures were prepared of cerebral cortex from neonate Wistar rats. Morphology was evaluated by phase-contrast microscopy and immunocytochemistry for glial fibrillary acidic protein (GFAP). Glutamate uptake was measured using l-[2,3-³H]glutamate. Glutamine synthetase and content of glutathione were measured by enzymatic colorimetric assays. S100B content was determined by ELISA. Typical polygonal morphology becomes stellated when astrocyte cultures were exposed to 250 μM resveratrol for 24 h. At concentration of 25 μM, resveratrol was able to increase glutamate uptake and glutathione content. Conversely, at 250 μM, resveratrol decreased glutamate uptake. Unexpectedly, resveratrol at this high concentration increased glutamine synthetase activity. Extracellular S100B increased from 50 μM upwards. Our findings reinforce the protective role of this compound in some brain disorders, particularly those involving glutamate toxicity. However, the underlying mechanisms of these changes are not clear at the moment and it is necessary caution with its administration because elevated levels of this compound could contribute to aggravate these conditions.     

Intracellular Mg<Superscript><Emphasis Type="Bold">2+</Emphasis></Superscript> Influences Both Open and Closed Times of a Native Ca<Superscript><Emphasis Type="Bold">2+</Emphasis></Superscript>-activated BK Channel in Cultured Human Renal Proximal Tubule Cells

Effects of intracellular Mg²⁺ on a native Ca²⁺-and voltage-sensitive large-conductance K⁺ channel in cultured human renal proximal tubule cells were examined with the patch-clamp technique in the inside-out mode. At an intracellular concentration of Ca²⁺ ([Ca²⁺]_i) of 10⁻⁵–10⁻⁴ M, addition of 1–10 mM Mg²⁺ increased the open probability (P_o) of the channel, which shifted the P_o –membrane potential (V_m) relationship to the negative voltage direction without causing an appreciable change in the gating charge (Boltzmann constant). However, the Mg²⁺-induced increase in P_o was suppressed at a relatively low [Ca²⁺]_i (10^−5.5–10⁻⁶ M). Dwell-time histograms have revealed that addition of Mg²⁺ mainly increased P_o by extending open times at 10⁻⁵ M Ca²⁺ and extending both open and closed times simultaneously at 10^−5.5 M Ca²⁺. Since our data showed that raising the [Ca²⁺]_i from 10⁻⁵ to 10⁻⁴ M increased P_o mainly by shortening the closed time, extension of the closed time at 10^−5.5 M Ca²⁺ would result from the Mg²⁺-inhibited Ca²⁺-dependent activation. At a constant V_m, adding Mg²⁺ enhanced the sigmoidicity of the P_o–[Ca²⁺]_i relationship with an increase in the Hill coefficient. These results suggest that the major action of Mg²⁺ on this channel is to elevate P_o by lengthening the open time, while extension of the closed time at a relatively low [Ca²⁺]_i results from a lowering of the sensitivity to Ca²⁺ of the channel by Mg²⁺, which causes the increase in the Hill coefficient. M. Kubokawa and Y. Sohma contributed equally to this work.     

Comparative study of effects of cadmium cations in free and chelated forms on activity of glutathione S-transferase, growth, and endocytosis in culture of the infusoriumTetrahymena pyriformis

Effects of cadmium cations in free (Cd²⁺) and chelated with EDTA (Cd²⁺-EDTA) forms were studied on growth, endocytosis, and activity of glutathione S-transferase (GT) in the free-living infusoriaTetrahymena pyriformis. It is shown that the cytotoxicity of Cd²⁺ in the free form at a concentration of 10 μM is much higher than of the Cd²⁺-EDTA complex at the equimolar concentration. Even at a low concentration (2 μM), Cd²⁺ produces an inhibition of the growth rate and endocytosis in theT. pyriformis culture, while the Cd²⁺-EDTA complex suppresses these functions insignificantly. Cd²⁺ in the free form at concentrations of 10 and 100 μM reduced activity of glutathione S-transferase by 39 and 61%. The chelated Cd²⁺-EDTA complex at these concentrations inhibited the GT activity by 5 and 55%, respectively.     

Effects of Cd2+ on the physiological state and photosynthetic activity of young barley plants

Barley plants (Hordeum vulgare L. cv. Obzor) were grown as a water culture in a climatic room. One part of them was subjected to a long-term Cd²⁺ stress - 12 d with 5.4×10⁻⁵ M Cd. The Cd²⁺ stress inhibited formation of the photosynthetic apparatus and its capacity for ¹⁴C photoassimilation, decreased the content of soluble proteins, increased the dark respiration rate and the free amino acids content, disturbed plant water relations, as well as the distribution of ¹⁴C within primary photoproducts of the treated barley plants. This revised version was published online in June 2006 with corrections to the Cover Date.     

Role of glutamine synthetase in citric acid fermentation byAspergillus niger

The activity of glutamine synthetase fromAspergillus niger was significantly lowered under conditions of citric acid fermentation. The intracellular pH of the organism as determined by bromophenol blue dye distribution and fluorescein diacetate uptake methods was relatively constant between 6·0–6·5, when the pH of the external medium was varied between 2·3–7·0.Aspergillus niger glutamine synthetase was rapidly inactivated under acidic pH conditions and Mn²⁺ ions partially protected the enzyme against this inactivation. Mn²⁺-dependent glutamine synthetase activity was higher at acidic pH (6·0) compared to Mg²⁺-supported activity. While the concentration of Mg²⁺ required to optimally activate glutamine synthetase at pH 6·0 was very high (≥ 50 mM), Mn²⁺ was effective at 4 mM. Higher concentrations of Mn²⁺ were inhibitory. The inhibition of both Mn²⁺ and Mg²⁺-dependent reactions by citrate, 2-oxoglutarate and ATP were probably due to their ability to chelate divalent ions rather than as regulatory molecules. This suggestion was supported by the observation that a metal ion chelator, EDTA also produced similar effects. Of the end-products of the pathway, only histidine, carbamyl phosphate, AMP and ADP inhibitedAspergillus niger glutamine synthetase. The inhibitions were more pronounced when Mn²⁺ was the metal ion activator and greater inhibition was observed at lower pH values. These results permit us to postulate that glutamine synthesis may be markedly inhibited when the fungus is grown under conditions suitable for citric acid production and this block may result in delinking carbon and nitrogen metabolism leading to acidogenesis