Effects of potassium on the ethanol production rate of Saccharomyces cerevisiae carrying the plasmid pCYG4 related with ammonia assimilation

The influence of potassium on ethanol production bySaccharomyces cerevisiae wild type and AR5 cells carrying the plasmid pCYG4 was investigated. This plasmid carries the glutamate dehydrogenase gene conferring an 11-fold higher level of expressed enzyme activity over the wild type cells. All experiments were carried out in batch culture with medium supplemented to different potassium concentrations up to 180 mM. Maximum ethanol production rate was observed in the AR5 cells grown in medium supplemented with 3.5 mM of potassium ions. Glucose uptake rate increased with increasing potassium up to 60 mM, but higher concentrations depressed glucose uptake rate in both strains. Furthermore, the wild type cells showed higher growth rate, ethanol production, and glucose consumption rate than the AR5 cells. These lower rates in the AR5 cells could be explained by repression of potassium uptake by an enhancement of ammonium feeding, and greater energy requirements by these cells due the presence of the plasmid.     

RESPIRATION RESPONSES OF A polA1 AND A tif-1 MUTANT OF ESCHERICHIA COLI TO FAR-ULTRAVIOLET IRRADIATION*

Abstract— Cessation of respiration in Escherichia coli 60min after far-ultraviolet (254 nm) irradiation is dependent upon the recA and lexA gene products and is regulated by cyclic 3′,5′-adenosine monophosphate (cAMP) and its receptor protein. Respiration responses to UV irradiation were studied in two E. coli B/r mutants, polA1 and tif-1, both of which express other rec/lex functions (such as mutagenesis) after UV irradiation. The cells were grown on glycerol minimal medium supplemented with required amino acids. After receiving a relatively high UV fluence, the polA1 mutant, deficient in DNA polymer-ase I, showed a respiration shutoff response like the wild type cells. 5-Fluorouracil and rifampin, an inhibitor of initiation of RNA synthesis, did not prevent respiration shutoff in the mutant cells as they did in the wild type cells. Thus, RNA synthesis is not necessary for cessation of respiration in polA1 cells and the process is not an induced one. At lower fluences which did not shut off respiration of polA1 cells, cAMP did not cause a more complete shutoff as it did for the wild type cells. The tif-1 mutant has a modified recA protein, and when unirradiated cells are incubated at 42°C they form filaments, mutate, and show other rec/lex responses. This mutant did not shut off its respiration at either 30 or 42°C, and the response was not modified by cAMP. An E. coli K12 strain, W3110, was also tested for its respiration response to UV. At 52J/m² respiration did not shut off and cAMP had no effect.     

Enhanced β-Galactosidase Production from Whey Powder by a Mutant of the Psychrotolerant Yeast <Emphasis Type="Italic">Guehomyces pullulans</Emphasis> 17<Emphasis Type="Italic">–</Emphasis>1 for Hydrolysis of Lactose

In order to isolate β-galactosidase overproducers of the psychrotolerant yeast Guehomyces pullulans 17–1, its cells were mutated by using nitrosoguanidine (NTG). One mutant (NTG-133) with enhanced β-galactosidase production was obtained. The mutant grown in the production medium with 30.0 g/l lactose and 2.0 g/l glucose could produce more β-galactosidase than the same mutant grown in the production medium with only 30.0 g/l lactose while β-galactosidase production by its wild type was sensitive to the presence of glucose in the medium. It was found that 40.0 g/l of the whey powder was the most suitable for β-galactosidase production by the mutant. After optimization of the medium and cultivation conditions, the mutant could produce 29.2 U/ml of total β-galactosidase activity within 132 h at the flask level while the mutant could produce 48.1 U/ml of total β-galactosidase activity within 144 h in 2-l fermentor. Over 77.1% of lactose in the whey powder (5.0% w/v) was hydrolyzed in the presence of the β-galactosidase activity of 280 U/g of lactose within 9 h while over 77.0% of lactose in the whey was hydrolyzed in the presence of β-galactosidase activity of 280 U/g of lactose within 6 h. This was the first time to show that the β-galactosidase produced by the psychrotolerant yeast could be used for hydrolysis of lactose in the whey powder and whey.     

Phosphotransacetylase as a key factor in biological production of polyhydroxybutyrate

Phosphotransa cetylase (Pta) catalyzes the reversible conversion of, acetyl-coenzyme A (CoA) to acetyl phosphate. Polyhydroxybutyrate (PHB) synthase and accumulation were compared between a Pta-deficient mutant and the wild-type Escherichia coli, which were transformed with pAE100, coding for 3-ketothiolase, NADPH-dependenta cetoacetyl-CoA reductase, and PHB synthase from Ralstonia eutropha. During the growth period, PHB synthase activity in the Pta-deficient mutant was lower than that in the wild type. PHB accumulation in the Pta-deficient mutant, however, was higher than that in wild-type cells grown in Luria-Bertani (LB) medium containing 1% glucose (high C:N ratio). The Pta-deficient mutant showed PHB accumulation even in LB medium (low C:N ratio), whereas wild-type cells showed no PHB accumulation. These data suggest the activation of PHB synthase by acetyl phosphate that is synthesized by Pta. A decrease in Pta activity probably causes some increase in acetyl-CoA as substrate for the PHB synthesis pathway, resulting in increased PHB accumulation.     

Effect of growth phase on the Escherichia coli response to ultraviolet-A radiation: influence of conditioned media, hydrogen peroxide and acetate

The results reported herein indicate that the ultraviolet-A (UVA) radiation-induced effects in Escherichia coli depend on its growth phase. Stationary-phase cells recover faster from a sub-lethal UVA exposure and have a higher resistance to lethal effect of the radiation than exponential growing cells. Although pre-incubation in spent medium supernatant increased the resistance of log-phase cells to lethal UVA effects, this pre-treatment considerably prolonged the duration of the radioinduced sub-lethal growth delay. The aim of the present study was to investigate the effect exerted by the E. coli conditioned media and evaluate the influence of nutritional stress, hydrogen peroxide and acetate. Pre-incubated in conditioned medium, cells in exponential growth phase were irradiated and the induced effects were compared with those found when catalase, high culture densities and acetate were employed. Unexpectedly, the duration of the growth delay in cells submitted to these treatments was shortened in comparison with control cells incubated in conditioned medium with no modifications. Lengthening of the growth delay was mimicked when exponentially growing cells were incubated in fresh medium supplied with 5 microM H(2)O(2). The effects of spent medium on wild type and rpoS mutant strains were similar, indicating that this response is independent of RpoS controlled functions. We assumed that an oxidative component of the spent medium, probably H(2)O(2), could be involved in the observed phenomenon. This effect is specific of E. coli and independent of rpoS.     

Effect of primary metabolites on protein utilization byAgaricus bisporus,Coprinus cinereus,andVolvariella volvacea

Protein is probably a major nitrogen constituent in the natural habitats of basidiomycete fungi. The ability of two commercial crop species, Agaricus bisporus and Volvariella volvacea, and a common weed, Coprinus cinereus, to degrade protein was investigated. Mycelia were incubated at 25‡C in static liquid cultures on completely defined media supplemented with 1% (w/v) soluble casein as the protein source. Glucose, ammonium, or sulfate were added as alternative sources of the major elements. Tests were also made on media devoid of protein. Alternative nitrogen or sulfur sources had little or no effect on the rate of protein degradation, pH, medium glucose, or ammonium concentrations or mycelial dry weight yields. However, addition of glucose affected these parameters markedly, with a 3–4-fold increase in the dry weights of all three organisms and a two-fold increase in the rate of protein degradation by C. cinereus and V. volvacea. A. bisporus degraded protein at the same rate in both the presence or absence of glucose. Growth of the organisms was similar whether glucose or protein was the sole source of carbon. With glucose present, the pH decreased during the early stages of growth, being correlated with glucose utilization. Upon glucose disappearance, the pH increased in the presence of protein, but no subsequent change occurred with the glucose as a sole carbon source. In the absence of protein, the pH decreased to pH 3. At this pH no subsequent changes occurred in dry weight yields, glucose, or ammonium. With protein present, changes in medium ammonium concentration were correlated with dry weight yield, protein utilization, and pH. Thus, a strong correlation appears to exist between protein and glucose utilization and changes in pH and ammonium as well as dry weight yield. The increase in medium ammonium is probably caused by the deamination of the protein in the absence of glucose and its utilization as an energy source. However, the protein is utilized as efficiently as is glucose as a sole source of carbon.     

Yields from glucose, xylose, and paper sludge hydrolysate during hydrogen production by the extreme thermophile Caldicellulosiruptor saccharolyticus

This study addressed the utilization of an industrial waste stream, paper sludge, as a renewable cheap feedstock for the fermentative production of hydrogen by the extreme thermophile Caldicellulosiruptor saccharolyticus. Hydrogen, acetate, and lactate were produced in medium in which paper sludge hydrolysate was added as the sole carbon and energy source and in control medium with the same concentration of analytical grade glucose and xylose. The hydrogen yield was dependent on lactate formation and varied between 50 and 94% of the theoretical maximum. The carbon balance in the medium with glucose and xylose was virtually 100%. The carbon balance was not complete in the paper sludge medium because the measurement of biomass was impaired owing to interfering components in the paper sludge hydrolysate. Nevertheless, >85% of the carbon could be accounted for in the products acetate and lactate. The maximal volumetric hydrogen production rate was 5 to 6 mmol/(L·h), which was lower than the production rate in media with glucose, xylose, or a combination of these sugars (9–11 mmol/[L·h]). The reduced hydrogen production rate suggests the presence of inhibiting components in paper sludge hydrolysate.     

Hyperthermostable,Ca<Superscript>2+</Superscript>-Independent,and High Maltose-Forming α-Amylase Production by an Extreme Thermophile <Emphasis Type="Italic">Geobacillus thermoleovorans</Emphasis>: Whole Cell Immobilization

The synthesis of extracellular α-amylase in Geobacillus thermoleovorans was constitutive. The enzyme was secreted in metabolizable carbon sources as well as non-metabolizable synthetic analogues of glucose, but the titers were higher in the former than that in the latter. G. thermoleovorans is a fast-growing facultatively anaerobic bacterium that grows under both aerobic and anaerobic conditions and produces an extracellular amylolytic enzyme α-amylase with the by-product of lactic acid. G. thermoleovorans is a rich source of various novel thermostable biocatalysts for different industrial applications. α-Amylase synthesis was subject to catabolite repression in the presence of high concentrations of glucose. The addition of cAMP to the medium containing glucose did not result in the repression of α-amylase synthesis. The addition of maltose (1%) to the starch arginine medium resulted in a twofold enhancement in enzyme titers. Polyurethane foam (PUF)-immobilized cells secreted α-amylase, which was higher than that with the free cells. PUF appeared to be a better matrix for immobilization of the thermophilic bacterium than the other commonly used matrices. The repeated use of PUF-immobilized cells was possible over 15 cycles with a sustained α-amylase secretion. The use of this enzyme in starch saccharification eliminates the addition of Ca²⁺ in starch liquefaction and its subsequent removal by ion exchangers from the product streams.     

Amperometric monitoring of redox activity in intact,permeabilised and lyophilised cells of the yeast Hansenula polymorpha

An effect of permeabilisation and lyophilisation of the yeast cells Hansenula polymorpha on their electrochemical behaviour in the presence of mediators, substrates (formaldehyde, glucose, methanol, ethanol), and cofactors (NAD⁺, NADP⁺, NADH, NADPH, glutathione) has been studied. Two amperometric techniques differing in the cell immobilisation methods were applied. The cells of a wild strain (356) and mutant strains (C-105 and KCA 33) of the yeast, grown in the presence of glucose or methanol, were used in the experiments. The intact cells revealed the highest reduction rates of mediators, 2,6-dichlorphenolindophenol (DCIP) and 2,4-benzoquinone (BQ), as measured by amperometry. The addition of formaldehyde significantly enhanced the response, if the cells were grown in the presence of glucose. The permeabilised cells showed the lowest current level in the presence of DCIP and BQ and no response to the addition of formaldehyde and NAD⁺. However, the addition of NADH gave significant current surge. All these phenomena imply that the permeabilised cells lost cofactors and the activity of dehydrogenases producing NADH, but they remained the activity of NADH-ubiquinone oxidoreductase and of some components of the electron transport chain. The electrochemical behaviour of the lyophilised cells shows they are heterogeneous. The partial degradation of the outer membrane of the cells after their lyophilisation was electrochemically confirmed.     

PHOTOINACTIVATION OF A Chlamydomonas MUTANT(NL–11) IN THE PRESENCE OF METHIONINE: ROLES OF H2O2 and O-2

Abstract— A mutant of Chlamydomonas reinhardtii (NL–11) isolated from a wild type (137c+) was inactivated in the light in the presence of methionine at concentrations where the wild type was not inactivated. The inactivation was suppressed by either catalase or superoxide dismutase (SOD). Light-induced H₂O₂ formation and nitroblue tetrazolium (NBT) reduction inNL–11 were greater than those in the wild type. Methionine stimulated both the H₂O₂ formation and the NBT reduction inNL–11 as well as the wild type. The light-induced NBT reduction inNL–11 in the presence of methionine was partially suppressed by externally added SOD suggesting the participation of O^-₂. These results suggest that the hypersensitivity ofNL–11 to methionine in the light is due to stimulated formation of H₂O₂ and O^-₂.     

PHOTOMOVEMENT IN AN "EYELESS" MUTANT OF Chlamydomonas

The function of the stigma ("eyespot") in the green flagellate Chlamydomonas reinhardtii was investigated by comparing the photomovement of the wild-type alga with that of an "eyeless" mutant ( ey 627). Movements of individual cells in response to a blue-green stimulus light were recorded using a videomicroscope system and were analyzed using vectorial methods. Cells of the "eyeless" mutant were phototactic; at a high stimulus fluence rate, their swimming paths were directed away from the light source. Although the orientation of the mutant was not as strongly directional as that of the wild type, it was statistically significant. However, the swimming paths of the mutant cells were very erratic in the presence of the stimulus beam, undergoing frequent changes of direction. Despite the differences in their phototactic orientation, cells of mutant and wild type all showed a distinct step-up photophobic response at the onset of stimulation. These results are consistent with the hypothesis that the stigma plays an accessory role in phototaxis, either by shading the photoreceptor or by acting as a quarter-wave reflector.     

RESPONSE OF THE WILD-TYPE and HIGH LIGHT-TOLERANT MUTANT OF Anacystis nidulans AGAINST PHOTOOXIDATIVE DAMAGE: DIFFERENTIAL MECHANISM OF HIGH LIGHT TOLERANCE

Abstract— A high light-tolerant mutant of Anacystis was able to tolerate about three-fold higher light energy irradiance (30 W m^-2) than the wild type (10 W m^-2). The loss of sulfhydryl content and rate of lipid peroxidation in the wild-type cells is lower than in the mutant cells at high light irradiance. This phenomenon in the wild type is probably due to high light-induced severe photoinhibitory conditions resulting in a decreased rate of O₂ evolution. Results on the bleaching of the N, N '-dimethyl- p -nitrosoaniline at high light irradiance show a higher rate of bleaching in the wild-type than in the mutant cells. Further, results on the rate of N, N '-dimethyl- p -nitrosoani)ine bleaching in the presence of radical scavengers like sodium azide, histidine and sodium formate (10 m M , each) suggest that singlet oxygen is the predominant oxygen species produced in both the wild-type and mutant cells under high light. However, a similar quenching effect of formate in the mutant cells is indicative of increased formation of hydroxyl radicals. This observation is further corroborated by higher rate of lipid peroxidation. In addition to this, the superoxide dismutase activity is higher in the mutant (1.2 unit) than in the wild type. Taken together, these results suggest that the cells of the high light-tolerant mutant have an efficient intracellular mechanism to transform the free oxygen radicals.     

Effect of substrate inhibition and cooperativity on the electrochemical responses of glucose dehydrogenase. Kinetic characterization of wild and mutant types

Thanks to its insensitivity to dioxygen and to its good catalytic reactivity, and in spite of its poor substrate selectivity, quinoprotein glucose dehydrogenase (PQQ-GDH) plays a prominent role among the redox enzymes that can be used for analytical purposes, such as glucose detection, enzyme-based bioaffinity assays, and the design of biofuel cells. A detailed kinetic analysis of the electrochemical catalytic responses, leading to an unambiguous characterization of each individual steps, seems a priori intractable in view of the interference, on top of the usual ping-pong mechanism, of substrate inhibition and of cooperativity effects between the two identical subunits of the enzyme. Based on simplifications suggested by extended knowledge previously acquired by standard homogeneous kinetics, it is shown that analysis of the catalytic responses obtained by means of electrochemical nondestructive techniques, such as cyclic voltammetry, with ferrocene methanol as a mediator, does allow a full characterization of all individual steps of the catalytic reaction, including substrate inhibition and cooperativity and, thus, allows to decipher the reason that makes the enzyme more efficient when the neighboring subunit is filled with a glucose molecule. As a first practical illustration of this electrochemical approach, comparison of the native enzyme responses with those of a mutant (in which the asparagine amino acid in position 428 has been replaced by a cysteine residue) allowed identification of the elementary steps that makes the mutant type more efficient than the wild type when cooperativity between the two subunits takes place, which is observed at large mediator and substrate concentrations. A route is thus opened to structure-reactivity relationships and therefore to mutagenesis strategies aiming at better performances in terms of catalytic responses and/or substrate selectivity.     

Detection of NAD+-dependent alcohol dehydrogenase activities in YR-1 strain of Mucor circinelloides, a potential bioremediator of petroleum contaminated soils

Different soluble NAD⁺-dependent alcohol dehydrogenase (ADH) isozymes were detected in cell-free homogenates from aerobically grown mycelia of YR-1 strain of Mucor circinelloides isolated from petroleumcontaminated soil samples. Depending on the carbon source present in the growth media, multiple NAD⁺-dependent ADHs were detected when hexadecane or decane was used as the sole carbon source in the culture media. ADH activities from aerobically or anaerobically grown mycelium or yeast cells, respectively, were detected when growth medium with glucose added was the sole carbon source; the enzyme activity exhibited optimum pH for the oxidation of different alcohols (methanol, ethanol, and hexadecanol) similar to that of the corresponding aldehyde (≈7.0). Zymogram analysis conducted with partially purified fractions of extracts from aerobic mycelium or anaerobic yeast cells of the YR-1 strain grown in glucose as the sole carbon source indicated the presence of a single NAD⁺-dependent ADH enzyme in each case, and the activity level was higher in the yeast cells. ADH enzyme from mycelium grown in different carbon sources showed high activity using ethanol as substrate, although higher activity was displayed when the cells were grown in hexadecane as the sole carbon source. Zymogram analysis with these extracts showed that this particular strain of M. circinelloides has four different isozymes with ADH activity and, interestingly, one of them, ADH4, was identified also as phenanthrene-diol-dehydrogenase, an enzyme that possibly participates in the aromatic hydrocarbon biodegradation pathway.     

ROLES OF URACIL-DNA GLYCOSYLASE AND APYRIMIDINIC ENDONUCLEASES IN THE MOLECULAR 5-BROMO-2'-DEOXYURIDINE PHOTOSENSITIZATION IN Escherichia coli K-12

The molecular mechanism for 5-bromo-2'-deoxyuridine (BrdU) photosensitization was studied in thymine-requiring wild-type and uracil-DNA glycosylase (UDG)-deficient ung mutant cells of Escherichia coli K-12. Wild-type cells were more sensitive to BrdU photosensitiLation than ung mutant cells. IJV induced the identica/ numbers of alkaline sucrose single-strand breaks (SSB) in 5-bromouracil-DNA (BrU-DNA) of both the wild type and ung mutant. The ung mutant cells repaired SSB almost completely, whereas the wild-type cells with UDG produced more adverse SSB by 90 min after UV. Neutral agarose gel electrophoresis of minipreps indicated that UV induced (1) more smears of host BrU-DNA possibly by more double-strand breaks (DSB) and (2) a greater decline of pBR322 Form I BrU-DNA in the wild-type cells than the ung cells. These results indicated a greater induction of SSB by apyrimidinic (AP) endonucleases in wild-type cells. The ung/ wild ratios (=1.7–1.9) for cellular and plasmid BrdU sensitizations aftcr growth in 50% BrdU were similar. The extents of UDG-dependent and UDG-independent sensitizations in wild-type cells were ∼40 and ∼60%, respectively. The xth nfo double mutant defective in both exonuclcase III and endonucleasc IV was more sensitive to BrdU photosensitization than the wild type, indicating that an excess of AP sites remaining after uracil excision in the xth nfo mutant causes a greater BrdU photosensitization than SSB by AP endonucleases in wild-type cells. Conversely, the xth rfo ung triple mutant was more resistant to BrdU photosensitization than the xth nfo double mutant, so that UV-induced uracil residues in the BrU-DNA are tolerated and do not appear to be directly responsible for BrdU photosensitization.     

Gap junctional intercellular communication (GJIC) in rat glioma cells--characterizations to detect inhibitors of metabolic cooperation

Gap junctional intercellular communication (GJIC) as measured by metabolic cooperation was examined in a rat glioma cell line P98F47. X-ray induced mutants of P98F47 cells were grown in 6-thioguanine selective medium (6TG medium) to separate 6TG-resistant HGPRT- mutant cells (6TGr). By co-culturing 200 6TGr cells with varied high densities of the wild type 6TG-sensitive cells (6TGs), it was found that the recovery of 6TGr cells depended on the density of 6TGs cells. Higher densities of 6TGs cells reduced the recovery of 6TGr cells. These results demonstrate the ability of P98F47 cells to perform metabolic cooperation which is indicative of GJIC. When metabolic cooperation was inhibited, increased recovery of 6TGr cells was observed. Presented results also demonstrate metabolic cooperation between P98F47 glioma cells and normal rat glial cells. Effect of tumor promoting chemicals on metabolic cooperation of P98F47 cells was studied. 3H-uridine nucleotide autoradiography technique was used to confirm the above observations. The results suggest that these cells may provide the basis for an in vitro assay specially to study brain tumor promoters and neurotoxins.     

PHOTODYNAMIC DAMAGE AND ITS REPAIR IN Vibrio cholerae

Abstract— Repair of photodynamic damage induced by acriflavine and visible light has been examined in three strains of Vibrio cholerae differing in their capabilities to repair ultraviolet (UV) light induced DN A damage. Excision repair deficient wild type cells of strain 154 are more sensitive to photodynamic treatment compared to repair proficient cells of strain 569B. However, no difference in their capabilities to repair of damage following photodynamic treatment can be detected. No single-strand breaks in the irradiated cell DNA are observed when the cell survival is more than 10%. Single-strand breaks observed at cell survival less than 5% are not dark repairable even in excision repair proficient wild type cells. Repair of membrane damage can partially account for the recovery observed at low doses. In contrast, radiation-sensitive mutant 569B_s cells which lack both excision and medium-dependent dark repair for UV-lesions are most efficient in repairing damage induced by photodynamic treatment.     

Pyrolysis of Glucose over Two Amphoteric Metal Oxides

The catalytic pyrolysis of glucose over amphoteric metal oxide, ZnO or γ-Al2O3, was studied comparatively with direct pyrolysis. The effects of catalyst to glucose ratio on the yields of pyrolytic products and on the chemical composition of the liquid products were discussed. Compared with the pyrolytic products of direct pyrolysis, the amount of residual char decreased, whereas the gas yield increased in the presence of the catalysts. The highest liquid yield over ZnO(49.5%) was obtained when the ratio of ...     

WAVELENGTH DEPENDENCE OF INACTIVATION and MUTAGENESIS IN HAPLOID YEAST CELLS OF DIFFERENT SENSITIVITIES

The wavelength dependence of inactivation and mutagenesis in the range between 254 and 313 nm was investigated in haploid cells of the yeast Saccharomyces cerevisiae. The action spectra for a wild type and an excision deficient mutant essentially corresponded to those for bacteria and mammalian cells. There were, however, minor differences. The ratio of mutagenic to lethal action decreased with increasing wavelength in the wild type, but remained constant in the excision deficient mutant. This is discussed in terms of error-prone and erro-free repair. For both inactivation and mutagensis, photorcactivability was lower with longer wavelengths in the wild type, but did not change in the excision deficient mutant. Implications for the role of pyrimidine dimers are considered.     

GAP JUNCTIONAL INTERCELLULAR COMMUNICATION (GJIC) IN RAT GLIOMA CELLS — CHARACTERIZATIONS TO DETECT INHIBITORS OF METABOLIC COOPERATION

Gap junctional intercellular communication (GJIC) as measured by metabolic cooperation was examined in a rat glioma cell line P_(98)F_(47). X-ray induced mutants of P_(98)F_(47) cells were grown in 6-thioguanine selective medium (6TG medium) to separate 6TG-resistant HGPRT- mutant cells (6TG~r). By co-culturing 200 6TG~r cells with varied high densities of the wild type 6TG-sensitive cells (6TG~s), it was found that the recovery of 6TG~r cells depended on the density of 6TG~s cells. Higher densities of 6TG~s cells reduced the recovery of 6TG~r cells. These results demonstrate the ability of P_(98)F_(47) cells to perform metabolic cooperation which is indicative of GJIC. When metabolic cooperation was inhibited, increased recovery of 6TG~r cells was observed. Presented results also demonstrate metabolic cooperation between P_(98)F_(47) glioma cells and normal rat glial cells. Effect of tumor promoting chemicals on metabolic cooperation of P_(98)F_(47) cells was studied.~3H-uridine nucleot de echnique was