Glycollate production and excretion by Alcaligenes eutrophus.

Autotrophic cultures of the facultative chemolithotroph Alcaligenes eutrophus have been found to excrete glycollate. This excretion was greatly stimulated by the incorporation of up to 20% (v/v) oxygen in the hydrogen used for gassing. The stimulatory effect of oxygen was prevented by the addition of 10% (v/v) CO2 to the gassing mixture. Glycollate excretion only in the presence of oxygen was increased by the addition of 2-pyridyl-hydroxymethane sulphonic acid (HPMS), an inhibitor of glycollate oxidation, indicating that glycollate formation itself was stimulated by oxygen. No glycollate excretion by cultures grown heterotrophically on pyruvate was detected, either in the absence or presence of HPMS, under heterotrophic or autotrophic cells showed phosphoglycollate phosphatase and glycollate oxidoreductase activities, which were considerably lower in extracts prepared from pyruvate- or fructose-grown (heterotrophic) cells. The increase in activity of both enzymes upon cell transfer from heterotrophic to autotrophic growth was prevented by chloramphenicol and resembled the induction of D-ribulose 1,5-diphosphate carboxylase under the same conditions.     

Hemoglobin-stimulated growth and antioxidant activities in cultured cotton cells.

Changes in cellular reactive oxygen scavenging enzymes were assessed in suspension-derived cells of cotton (Gossypium herbaceum) cv. Dhumad following culture with a commercial bovine hemoglobin (Hb) solution (Erythrogen) at 1:100-1:1000 (v:v). Mean (+/- SEM) fresh (f.wt.) and dry weights (d.wt.) of cells after 25 d of culture were significantly (p <.05) greater in medium supplemented with 1:750 and 1:1000 (v:v) Erythrogen, compared to controls lacking Erythrogen. For example, with 1:750 (v:v) Erythrogen, mean cell f.wt. and d.wt. were increased by 45 and 31%, respectively. Total soluble cellular protein increased by 141, 176, and 191% with Erythrogen at 1:50, 1:750, and 1:1000 (v:v), respectively. Cellular catalase and glutathione reductase activities decreased significantly (p <.05) following addition of low concentrations (1:1000 and 1:750 v:v) of Erythrogen to culture medium. However, increasing the concentration of Erythrogen to a maximum of 1:100 (v:v), caused a concomitant increase in catalase to a maximum of 62% over control. Mean total superoxide dismutase activity increased linearly with increasing Erythrogen concentration, reaching a maximum mean value over 2-fold greater than control with 1:100 (v:v) Erythrogen. A similar trend was observed in cellular H2O2 content, which reached a maximum of 98% over control with 1:250 (v:v) Erythrogen. These results demonstrate that culture of cotton cells with Hb solution causes changes in cellular oxygenation sufficient to modify cellular antioxidant status.     

Effect of u.v. light irradiation, starvation and heat on Escherichia coliββ-D-galactosidase activity and other potential viability parameters

The effect of u.v. light irradiation and two other types of stress (heat and starvation) on cellular functions of Escherichia coli have been studied. The severe reduction of the culturable cell number (cfu) and the direct viable count (DVC) after exposure to moderate u.v. light doses (48 mWs cm-2), was not reflected by the dehydrogenase activity (5-cyano-2,3-ditolyl tetrazolium chloride (CTC)-positive cells), the membrane integrity (SYTOX Green-negative cells), the membrane potential (bis-(1,3-dibutylbarbituric acid) trimethine oxonol (DiBAC4[3]) (OXONOL)-negative cells), and the beta-D-galactosidase activity. All parameters were affected by high u.v. light doses. Cellular activities (CTC, SYTOX, OXONOL, beta-D-galactosidase activity) were intact in non-culturable cells with presumably severe damage to DNA, and the activities seemed not to be appropriate for detection of viable E. coli after u.v. light irradiation. Heating for 20-30 min at 63 degrees C was required to cause a severe loss of the beta-D-galactosidase activity and the numbers of CTC-positive, SYTOX Green-negative or OXONOL-negative cells. A large portion (> or = 38%) of pre-irradiated (190 mWs cm-2) cells maintained their ability to reduce CTC and exclude SYTOX Green and OXONOL after 51 d of starvation (dark, 7 degrees C) in phosphate-buffered saline.     

Correlation between calmodulin-like protein, phospholipids, and growth in glucose-grown Mycobacterium phlei.

In Mycobacterium phlei TMC 1548 supplementation of growth medium containing 2% v/v glycerol with glucose (up to 5% w/v) resulted in an increase in growth (yield of cells), in amount of total phospholipids, and in each of the individual phospholipids (cardiolipin, phosphatidylethanolamine, phosphatidylinositol and its mannosides, and phosphatidylglycerol). However, when the medium was supplemented with a higher concentration (7.5% w/v) of glucose, both growth and phospholipid levels decreased to near control values (2% v/v glycerol alone). Cyclic AMP levels, which decreased at all concentrations of glucose, had no relation to phospholipid content or growth. The presence of a protein that possesses the property of stimulating c-AMP phosphodiesterase activity was recently demonstrated in Mycobacterium smegmatis (Falah et al. 1988. FEMS Microbiol. Lett. 56: 89-93). In M. phlei the level of this calmodulin-like protein (assayed by radioimmunoassay) changed with different concentrations of glucose in the growth medium in a manner identical with that of phospholipids. We suggest that in mycobacteria (i) intracellular calmodulin-like protein levels are affected by glucose concentration in the growth medium and (ii) there is a positive correlation between the levels of calmodulin-like protein, total and individual phospholipids, and growth (yield of cells) in glucose-grown M. phlei.     

Alpha-V-dependent outside-in signaling is required for the regulation of CD44 surface expression, MMP-2 secretion, and cell migration by osteopontin in human melanoma cells

The level of integrin alpha(v)beta3 and its ligand osteopontin (OPN) has been directly correlated to tumorigenicity of melanoma and other cancer cells. We have previously shown an increase in pp(60c-Src) kinase activity associated with integrin alpha(v)beta3 in melanoma cells (M21) treated with soluble OPN. pp(60c-Src) kinase activity was not observed in melanoma cells expressing alpha(v) that lacks the cytoplasmic domain (alpha(v)995). Results of the current study demonstrate that the amino acid sequence '995RPPQEEQERE1004' in the beta-turn of alpha(v) chain is required for the interaction of pp(60c-Src). Our results suggest that the beta-turn of alpha(v) chain may be indispensable for alpha(v)-associated signaling complex formation and outside-in signaling. To further analyze the alpha(v)beta3 signaling in melanoma cells, we over expressed OPN in M21 cells (M21/OPN). CD44 surface expression and MMP-2 activity in the conditioned medium were increased to a greater extent in M21/OPN cells as compared with M21 or alpha(v)995 cells. Also, M21/OPN cells exhibit increased motility, which is markedly reduced upon treatment with inhibitors to alpha(v) and MMP-2. Our findings suggest that the increase in MMP-2 activity is integrin-dependent as MMP-2 activity is reduced in cells treated with an inhibitor to alpha(v) or in alpha(v)995 cells expressing mutant alpha(v).     

In vitro assessment of a chemically synthesized Shiga toxin receptor analog attached to chromosorb P (Synsorb Pk) as a specific absorbing agent of Shiga toxin 1 and 2.

A synthetic analog of Shiga toxin (Stx) receptor (Synsorb Pk) was quantitatively assessed to determine whether it can protect human renal adenocarcinoma cells (ACHN cells) from the cytotoxicity of Stx1 and Stx2 by coincubation experiments. Coincubation of 100 and 20 ng of Stxl and Stx2 with 50 mg of Synsorb Pk for 1 hr at 37 C in 1 ml of Eagle's Minimum Essential Medium supplemented with 1% (v/v) non-essential amino acid and 10% (v/v) fetal calf serum protected 50% of the cells from the cytotoxic effect. Chromosorb P, an inert matrix control, did not absorb the Stxs at all. Heat-treatment (boiled for 10 min) to Synsorb Pk caused a 50% decrease in Stx2-binding activity, but did not effect the Stx1 binding. Further, Stxs bound to Synsorb Pk could be demonstrated. When 20 mg of Synsorb Pk was coincubated for 30 min at 37 C in 1 ml of phosphate-buffered saline with 1 and 10 ng or more of Stx1 or Stx2, respectively, the toxins could be detected on the surface when the bound toxins on Synsorb Pk were used as the solid phase in enzyme immunoassay. The amount of 100 ng/ml of both Stxl and Stx2 appeared to saturate 20 mg/ml of Synsorb Pk after coincubating for 30 min at 37 C. While assessing the Stxs' binding activity to Synsorb Pk, it was demonstrated that Stxl had a higher affinity to Pk trisaccharide than Stx2. These observations provide useful information on the effectiveness of Synsorb Pk to trap and eliminate free Stxs produced in the gut of patients infected by Stx-producing Escherichia coli, and to prevent the progression of hemorrhagic colitis to hemolytic uremic syndrome.     

Immobilization of permeabilized whole cell penicillin G acylase from Alcaligenes faecalis using pore matrix crosslinked with glutaraldehyde

The activity of penicillin G acylase from Alcaligenes faecalis increased 7.5-fold when cells were permeabilized with 0.3% (w/v) CTAB. The treated cells were entrapped by polyvinyl alcohol crosslinked with boric acid, and crosslinked with 2% (v/v) glutaraldehyde to increase the stability. The conversion yield of penicillin G to 6-aminopenicillanic acid was 75% by immobilized system in batch reaction. No activity was lost after 15 cycles and about 65% enzyme activity was retained at the end of the 31th cycle.     

Lactosucrose bioconversion from lactose and sucrose by whole cells of Paenibacillus polymyxa harboring levansucrase activity

Lactosucrose, a functional trisaccharide, was produced from lactose as an acceptor and sucrose as a fructosyl donor by whole cells harboring transfructosylation activity of levansucrase. Levansucrase-induced cells of Paenibacillus polymyxa were obtained in the medium containing sucrose, and the transfructosylation activity in the whole cell was optimized for lactosucrose production. The optimal cell concentration, substrates ratio, temperature, and pH were 2.0% (w/v), 22.5% (w/v) lactose and 22.5% (w/v) sucrose, 55 degrees C, and 6.0, respectively. Under these conditions, the whole cells produced approximately 17.0% (w/v) lactosucrose in 6 h of reaction time with a productivity of 2.8% (w/v)/h.     

Evaluation of cell viability and apoptosis in human amniotic fluid-derived stem cells with natural cryoprotectants

A previous study demonstrated that disaccharides, antioxidants, and caspase inhibitors can be used in freezing solutions to reduce the concentration of Me₂SO from the current standard of 10% (v/v) to 5% (v/v) or 2.5% and to eliminate fetal bovine serum (FBS) for the cryopreservation of human amniotic fluid-derived stem cells (AFSCs). Hence, this study investigated whether an irreversible inhibitor of caspase enzymes, benzyloxycarbonyl-Val-Ala-dl-Asp-fluoromethylketone (zVAD-fmk), could be used in post-thaw culture media to increase the survival rate of AFSCs. Our results showed that AFSCs cryopreserved in freezing solution containing trehalose, catalase, and 5% (v/v) Me₂SO and then supplemented with zVAD-fmk in the post-thaw culture media showed similar post-thawing viability, proliferation, and apoptosis than cells cryopreserved in the control solution (10% (v/v) Me₂SO and 20% FBS). The caspase-3 activity in all the cryopreservation solutions tested was similar to that of the control. Caspase-3, caspase-8, caspase-9, and PARP expression was not found in the cryopreserved cells. In addition, no difference was found in the survival rate and apoptosis between short-term (3 weeks) and long-term (1 year) storage of AFSCs cryopreserved in the solutions used in this study. The results of the present study demonstrate that recovery of cryopreserved cells was enhanced by using a caspase inhibitor in the post-thaw culture media.     

Differentiation of U937 cells enables a phospholipase D-dependent pathway of cytosolic phospholipase A2 activation.

Treatment with dibutyryl cyclic AMP (dBcAMP) of the human, premonocytic U937 cell line results in differentiation toward a monocyte/granulocyte-like cell. This differentiation enables the cell to activate cytosolic phospholipase A2 (cPLA2) to release arachidonate upon stimulation. In contrast, undifferentiated cells are unable to release arachidonate even when stimulated with calcium ionophores. In the present research, a role for phospholipase D (PLD) in the regulation of cPLA2 was shown based on a number of observations. First, the ionomycin- and fMLP-stimulated production of arachidonate in differentiated cells was sensitive to ethanol (2% (v/v)). Ethanol acts as an alternate substrate in place of water for PLD producing phosphatidylethanol (PEt) instead of phosphatidic acid. Indeed, ionomycin stimulation of differentiated cells produced a 14-fold increase in PEt levels. Further evidence for the involvement of PLD in the regulation of cPLA2 came from the observation that the stimulated production of diacylglycerol (for which phosphatidic acid is a major source) was greatly diminished in undifferentiated cells as compared to differentiated cells. Moreover, the normally deficient activation of cPLA2 in undifferentiated cells could be stimulated to release arachidonate if the cells were electroporated in the presence of GTP[gamma]S and MgATP. This treatment stimulates phosphatidylinositol-4,5-bisphosphate (PIP2) production which appears to activate PLD and cPLA2 in subsequent steps. The phosphatidic acid (and diacylglycerol derived from phosphatidic acid) appears to greatly regulate the action of cPLA2 by an unknown mechanism, and undifferentiated cells lack the ability to stimulate PLD activity due to a dysfunction of PIP2 production.     

Interleukin-2-induced decrease in the buoyant density of cytotoxic cells and its relationship with proliferation

Interleukin 2 (IL-2) is known to induce an augmentation of natural killer activity. In the present study we have used discontinuous Percoll density gradients to investigate the changes in the buoyant density of killer effector cells generated in response to IL-2. In all systems examined (mouse and rat spleen cells and human peripheral blood mononuclear cells), the cytolytic effectors generated in response to IL-2 have markedly lower buoyant densities compared to fresh natural killer cells. Our results also suggest that if a single layer of 44.2% (v/v) Percoll is used, almost all IL-2-induced cytolytic activity can be enriched in the cells which float on this layer whereas the heavier cells from the pellet are devoid of cytotoxic activity. The contribution of proliferative activity to (a) the generation of cytotoxicity and (b) the decrease in the buoyant density of the IL-2-induced killer cells was also studied in the mouse system. Natural killer levels in mouse spleen cells treated with mitomycin C could be significantly augmented by IL-2. Moreover, the effector cells generated in control as well as mitomycin-C-treated spleen cells in response to IL-2 had the same low buoyant densities. These results indicate that proliferation is not a prerequisite for the activation of killer activity and a reduction in buoyant density in response to IL-2.     

Specific esterase activity induced in mouse Friend erythroleukemia cells by dimethylsulfoxide.

Three cell lines of mouse erythroleukemia transformed by Friend virus (FLC), namely 745, F4-1, and 3BM-78, were grown for six days in the absence or in the presence of 1.5% (v/v) dimethylsulfoxide (DMSO) and compared cytochemically for naphtol-AS D-chloroacetate esterase (E), alkalinephosphatase (AP), myeloperoxidase (MP) and periodic acid Schiff (PAS) reaction activity. In the absence of inducer only 1–2% of slightly E positive cells could be found. E positivity greatly increased in 3BM-78 and F4-1 but poorly in 745 cells, after treatment with DMSO. Unlike E reaction, AP and MP reactions were positive in about 5% 3BM-78 and F4-1 cells without DMSO, but there were no positive cells after DMSO treatment. All three lines were always PAS negative. Hemoglobin synthesis (benzidine staining) was intensively induced by DMSO in all three lines. Morphologically after DMSO treatment, FLC matured displaying characteristics of basophilic megaloblastoid cells. The emergence of specific esterase activity, a marker of granulocytes, in FLC differentiating along the erythroid pathway, suggests that in these leukemia cells the genetic determinants for leukopoietic differentiation are retained and capable of being expressed phenotypically.     

The Low CO2-Inducible 36-Kilodalton Protein Is Localized to the Chloroplast Envelope of Chlamydomonas reinhardtii

The localization of the 36-kD polypeptide of Chlamydomonas reinhardtii induced by photoautotrophic growth on low CO2 concentrations (0.03% in air [v/v], low CO2-grown cells) has been investigated. This polypeptide was specifically localized to the chloroplast envelope membranes isolated from low CO2-grown cells and was not present in the chloroplast envelopes isolated from high (5% CO2 in air [v/v]) CO2-grown cells. The 36-kD protein does not show carbonic anhydrase activity and was not present on the plasma membranes isolated from low CO2-grown cells. This protein may, in part, account for the different inorganic carbon uptake characteristics observed in chloroplasts isolated from high and low CO2-grown cells of C. reinhardtii.     

Bioconversion of benzonitrile to benzoic acid using free and agar entrapped cells of Nocardia globerula NHB-2

The free and agar immobilized cells of Nocardia globerula NHB-2 having nitrilase (EC 3.5.5.1) activity were used to catalyse the transformation of benzonitrile to benzoic acid. The whole cells of N. globerula NHB-2 were immobilized in agar which exhibited maximum conversion of benzonitrile to benzoic acid in 0.1 M potassium phosphate buffer pH 7.5 (free cells) 8.0 (immobilized cells), temperature 40 degrees C, cells 2 mg dcm ml(-1) reaction mixture and benzonitrile (4% v/v) in 4 h (free cells). The effect of temperature on the stability of nitrilase was studied and cells retained 100% activity at 30 degrees C and lost 50% activity at 40 degrees C. In a fed batch mode of reaction 108 and 84 gl(-1) benzoic acid was produced using free and agar entrapped cells (2 g dcm). The agar immobilized cells were recycled up to three times and 80, 62, 20 gl(-1) benzoic acid was again produced respectively in each of three cycles and a total 244 g benzoic acid was produced by recycling the same mass of immobilized biocatalyst.     

Molecular identification and functional role of voltage-gated sodium channels in rat carotid body chemoreceptor cells. Regulation of expression by chronic hypoxia in vivo

We have assessed the expression, molecular identification and functional role of Na+ channels (Na(v)) in carotid bodies (CB) obtained from normoxic and chronically hypoxic adult rats. Veratridine evoked release of catecholamines (CA) from an in vitro preparation of intact CBs obtained from normoxic animals, the response being Ca2+ and Na+-dependent and sensitive to tetrodotoxin (TTX). TTX inhibited by 25-50% the CA release response evoked by graded hypoxia. Immunoblot assays demonstrated the presence of Na(v)alpha-subunit (c. 220 kDa) in crude homogenates from rat CBs, being evident an up-regulation (60%) of this protein in the CBs obtained from chronically hypoxic rats (10% O2; 7 days). This up-regulation was accompanied by an enhanced TTX-sensitive release response to veratridine, and by an enhanced ventilatory response to acute hypoxic stimuli. RT-PCR studies demonstrated the expression of mRNA for Na(v)1.1, Na(v)1.2, Na(v)1.3, Na(v)1.6 and Na(v)1.7 isoforms. At least three isoforms, Na(v)1.1, Na(v)1.3 and Na(v)1.6 co-localized with tyrosine hydroxylase in all chemoreceptor cells. RT-PCR and immunocytochemistry indicated that Na(v)1.1 isoform was up-regulated by chronic hypoxia in chemoreceptor cells. We conclude that Na(v) up-regulation represents an adaptive mechanism to increase chemoreceptor sensitivity during acclimatization to sustained hypoxia as evidenced by enhanced ventilatory responses to acute hypoxic tests.     

Bioconversion of butyronitrile to butyramide using whole cells of <Emphasis Type="Italic">Rhodococcus rhodochrous</Emphasis> PA-34

Butyramide is an important chemical commodity, which is used for the synthesis of hydroxamic acids and electrorheological fluids and for the preparation of β-amodoorganotin compounds. The nitrile hydratase (Nhase) of Rhodococcus rhodochrous PA-34 catalyzed the conversion of butyronitrile to butyramide. The maximum Nhase activity [18 U/mg dry cell weight (dcw)] of whole cells of R. rhodochrous PA-34 was observed at pH 7.0 with 10% (v/v) butyronitrile and 1 mg cells (dcw)/ml reaction mixture at 10°C. The cells of R. rhodochrous PA-34 retained almost 50% activity when incubated for 1 h in the presence of 85% (v/v) butyronitrile. A yield of 597 g of butyramide (6.8 M) was obtained using 60% (v/v) butyronitrile, 1 g cells (dry weight) in a 1-l batch reaction at 10°C for 6 h.     

Phorbol ester-like action of staurosporine on the cAMP response to prostaglandin E2 in two macrophage-like cell lines at distinct differentiation stages.

The purpose of this study is to investigate the involvement of protein kinase C (PKC) in prostaglandin E2 (PGE2)-stimulated cAMP production of two macrophage-like cell lines (G3 and XC). XC cells are thought to be placed at a more differentiated stage than G3 cells [Orikasa et al. (1991) Cell Immunol. 132, 350-365]. In RPMI 1640 containing 10% (v/v) heat-inactivated foetal calf serum (FCS), in which the cAMP response of both cells to PGE2 increased with duration of culture, XC cells showed greater response than G3 cells at 2 days of culture. In alpha-minimum essential medium (alpha-MEM) containing 20% (v/v) heat-inactivated horse serum (HS), the cAMP response of both cells was apparently greater than that in RPMI 1640 containing 10% (v/v) FCS. These cells increased cAMP production also in response to PGE1 and PGF2 alpha, and the order of potency in increase was PGE1 > PGE2 > PGF2 alpha. Interestingly, a short-term (20 min) treatment with phorbol 12-myristate 13-acetate (PMA), a direct activator of PKC or staurosporine, a relatively specific inhibitor of PKC, augmented the PGE2-stimulated cAMP production in these cells cultured in alpha-MEM containing 20% (v/v) HS. However, a long-term (24 h) treatment with these compounds did not alter the cAMP response. In G3 cells, PMA appeared more potent than staurosporine in terms of augmentation, whereas in XC cells, the former appeared less potent than the latter.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cryopreservation of Plasmodium chabaudi. II. Cooling and warming rates

Various cooling and warming rates were investigated to determine the optimum conditions for cryopreserving the intraerythrocytic stages of Plasmodium chabaudi. Infected blood, equilibrated in 10% v/v glycerol at 37 degrees C or in 15% v/v Me2SO at 0 degree C for 10 min, was cryopreserved using cooling rates between 1 and 5100 degrees C min-1. After overnight storage in liquid nitrogen the samples were warmed at 12,000 degrees C min-1. Warming rates between 1 and 12,000 degrees C min-1 were investigated using samples previously cooled at 3600 degrees C min-1. After thawing, the glycerol and Me2SO were removed by dilution in 15% v/v glucose-supplemented phosphate-buffered saline. Survival was assayed by inoculation of groups of five mice each with 10(6) infected cells and the time taken to reach a level of 2% parasitemia estimated. The optimum cooling rate was 3600 degrees C min-1 for parasites frozen using either 10% glycerol or 15% Me2SO; the pre-2% patent periods were 0.90 and 1.01 days above control values (representing survival levels of 21 and 17.5%, respectively). The optimum warming rate was 12,000 degrees C min-1; the pre-2% patent periods were 1.01 and 1.32 days above control values, respectively (18 and 10% survival), for glycerol and Me2SO. With ethanediol (5% v/v) and sucrose (15% w/v) as cryoprotectants the optimum warming rates were also 12,000 degrees C min-1 while the optimum cooling rates were 330 and 3600 degrees C min-1, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)