The influence of iron,sulphite and paraformaldehyde on the formation of 2.3 butanediol in the cultures of bacillus polymyxa

Summary InB. polymyxa cultures containing sucrose as the substrate the amount of 2.3 butanediol decreases between 10 and 30 days of fermentation but after 30 days, the formation of the glycol increases. If iron or sulphite ions are present in the culture ofB. polymyxa the yield of 2.3 butanediol is small in the first 10 days, as compared to the control culture but it gradually increases with the increase of the period of fermentation. This increase was observed upto 50 days. In the culture containing paraformaldehyde 2.3 butanediol is not formed. The sugar consumption in the cultures is high only during the first ten days of fermentation and after that though there is enough sugar present, it is not utilised and the amount of 2.3 butanediol goes on increasing. It appears that 2.3 butanediol is formed from some intermediate material formed during the period of high sugar consumption. Later the increase of 2.3 butanediol is due to its formation from the intermediate compound.     

The influence of minerals on the formation of 2,3 butanediol by Aerobacter aerogenes

Summary The presence of a small concentration of nickel or zinc ions in the cultures ofAerobacter aerogenes increases the formation of 2,3 butanediol in the cultures. The presence of 1 per cent of milk increases the formation of 2,3 butanediol in the cultures and decreases the consumption of sugar. Copper ions inhibit the formation of 2,3 butanediol.The combination of zinc and nickel ions decrease the formation of 2,3 butanediol in the cultures ofAerobacter aerogenes and even with addition of milk this combination is not beneficial to the formation of the diol. Nickel in presence of milk increases the formation of 2,3 butanediol.     

Zn++ stimulation of the induction of alcohol dehydrogenase by anaerobiosis in roots of Zea mays L.

Summary Zn⁺⁺ at an optimum concentration of 5×10^–4 M caused a two fold stimulation in the level of alcohol dehydrogenase (ADH) induced by anaerobic conditions. Isozymes specified by different genes and alleles show disproportionate increases in activity, such that, unequal representation of gene products while not eliminated, is invariably reduced by Zn⁺⁺ treatment. Thus in the case of alleles at the Adh-1 locus, there was a greater increase in the protein subunit specified by the Adh-1S allele. From previous work (Fischer and Schwartz, 1973) this protein is known to have a reduced affinity for Zn⁺⁺. This suggests that zinc availability during ADH induction is limiting and may provide an alternative to the cis-linked regulatory gene model proposed by Schwartz (1971) to explain the unequal expression of genes and alleles.     

CHARACTERIZATION OF LYMPHOCYTE TRANSFORMATION INDUCED BY ZINC IONS

Lymphocyte cultures from all normal human adults are stimulated by zinc ions to increase DNA and RNA synthesis and undergo blast transformation. Optimal stimulation occurs at 0.1 mM Zn⁺⁺. Examination of the effects of other divalent cations reveals that 0.01 mM Hg⁺⁺ also stimulates lymphocyte DNA synthesis. Ca⁺⁺ and Mg⁺⁺ do not affect DNA synthesis in this culture system, while Mn⁺⁺, Co⁺⁺, Cd⁺⁺, Cu⁺⁺, and Ni⁺⁺ at concentrations of 10^-7–10^-3 M are inhibitory. DNA and RNA synthesis and blast transformation begin to increase after cultures are incubated for 2–3 days with Zn⁺⁺ and these processes reach a maximum rate after 6 days. The increase in Zn⁺⁺-stimulated lymphocyte DNA synthesis is prevented by rendering cells incapable of DNA-dependent RNA synthesis with actinomycin D or by blocking protein synthesis with cycloheximide or puromycin. Zn⁺⁺-stimulated DNA synthesis is also partially inhibited by 5'-AMP and chloramphenicol. Zn⁺⁺ must be present for the entire 6-day culture period to produce maximum stimulation of DNA synthesis. In contrast to its ability to independently stimulate DNA synthesis, 0.1 mM Zn⁺⁺ inhibits DNA synthesis in phytohemagglutinin-stimulated lymphocytes and L1210 lymphoblasts.     

Studies on the mechanism of hrmonal stimulation of zinc uptake in human cell cultures: hormone-cell interactions and characteristics of zinc accumulation

Adrenal steroid hormones with glucocorticoid activity increase the uptake of Zn⁺⁺ in HeLa cell cultures. On the basis of the level of Zn⁺⁺ accumulation induced, steroid hormones can be classified into four groups: (a) optimal inducers (e.g., hydrocortisone and prednisolone); (b) suboptimal inducers (e.g., aldosterone and corticosterone); (c) anti-inducers (e.g., progesterone and 17 α-methyl testosterone) which competitively inhibit induction by optimal inducers; and (d) non-inducers (e.g., cortisone and pregnenolone) which neither induce nor inhibit the steroid-mediated increase in Zn⁺⁺ uptake. The ability of an anti-inducer to block the effects of optimal inducers is not the result of inhibition of steroid uptake or an effect on general protein synthesis. Optimal inducers do not increase adenyl cyclase activity of HeLa cells nor can the hormone effects on Zn⁺⁺ uptake be reproduced by 3'-5' cyclic AMP. The prednisolone-induced enhancement of Zn⁺⁺ uptake is gradually lost over two or three days following removal of the hormone. Uptake of Zn⁺⁺ by HeLa cells is not altered by a decrease of sodium concentration in the medium nor by changes in medium osmolarity. The uptake mechanism is not affected by subjecting intact cells to proteolytic enzymes; however, if cells are disrupted the hormone-mediated increase in Zn⁺⁺ accumulation is lost. The Zn⁺⁺ taken up by HeLa cells in the presence or absence of hormone is primarily cytoplasmic in localization and appears to be distributed in a multicompartmental system.     

Effect of zinc administration on thyrotropin releasing hormone-stimulated prolactinemia in healthy men

Previous in vitro studies have demonstrated zinc (Zn⁺⁺) inhibition of basal and of potassium (K⁺) or thyrotropin-releasing hormone (TRH)-stimulated prolactin (PRL) secretion, in a selective, reversible, and dose-dependent manner. Thus, Zn⁺⁺ may regulate physiologically pituitary PRL secretion. Furthermore, studies with patients with uremia, cirrhosis or prolactinoma, have shown the coexistence of hypozincemia and hyperprolactinemia and zinc supplementation did not correct hyperprolactinemia in these patients. In normal individuals Zn⁺⁺ administration produced controversial results on PRL secretion. Here, we investigated whether zinc administration affects TRH-stimulated PRL in healthy men. We found that Zn⁺⁺ administration does not change the TRH-stimulated PRL. Therefore, in normal conditions, Zn⁺⁺ does not inhibit TRH-stimulated prolactinemia. In addition, we found that acute increases of blood PRL and TRH do not alter blood Zn⁺⁺ levels.     

Effects of calcium and salinity on the growth rate of Ephydatia fluviatilis (Porifera: Spongillidae)

The freshwater sponge, Ephydatia fluviatilis (Porifera: Spongillidae), was maintained in a continuous-flow laboratory culture system under several conditions of calcium ion (Ca⁺⁺) concentration and salinity. Experimental results suggest that sponge growth rate increases with increasing Ca⁺⁺ concentration, that sponge growth rate decreases with increasing salinity, and that the negative effect of higher salinity can be overcome by increasing Ca⁺⁺ concentration. The experimental results correlate well with field observations on the effects of salinity and Ca⁺⁺ on the distribution of E. fluviatilis.     

Artificial consortium that produces riboflavin regulates distribution of acetoin and 2,3‐butanediol by Paenibacillus polymyxa CJX518

The introduction of an NADH/NAD⁺ regeneration system can regulate the distribution between acetoin and 2,3‐butanediol. NADH regeneration can also enhance butanol production in coculture fermentation. In this work, a novel artificial consortium of Paenibacillus polymyxa CJX518 and recombinant Escherichia coli LS02T that produces riboflavin (VB₂) was used to regulate the NADH/NAD⁺ ratio and, consequently, the distribution of acetoin and 2,3‐butanediol by P. polymyxa. Compared with a pure culture of P. polymyxa, the level of acetoin was increased 76.7% in the P. polymyxa and recombinant E. coli coculture. Meanwhile, the maximum production and yield of acetoin in an artificial consortium with fed‐batch fermentation were 57.2 g/L and 0.4 g/g glucose, respectively. Additionally, the VB₂ production of recombinant E. coli could maintain a relatively low NADH/NAD⁺ ratio by changing NADH dehydrogenase activity. It was also found that 2,3‐butanediol dehydrogenase activity was enhanced and improved acetoin production by the addition of exogenous VB₂ or by being in the artificial consortium that produces VB₂. These results illustrate that the coculture of P. polymyxa and recombinant E. coli has enormous potential to improve acetoin production. It was also a novel strategy to regulate the NADH/NAD⁺ ratio to improve the acetoin production of P. polymyxa.     

Stimulation of DNA synthesis and 2-deoxy-D-glucose transport in chick embryo cultures by excessive metal concentrations and by a carcinogenic hydrocarbon

Chick embryo cultures deprived of serum synthesize DNA at a reduced rate. DNA synthesis in serum-deprived cultures is stimulated as much as ten-fold by the addition of Zn⁺⁺, Mn⁺⁺ or Cd⁺⁺ in concentrations just below the toxic level. These metals, in the same concentration range, also stimulate the uptake of ³H-2-deoxy-D-glucose (2-DOG). The increase in uptake of 2-DOG precedes the increase in synthesis of DNA, and is probably an indicator of a more general membrane perturbation. The metals also stimulate DNA synthesis in serum-containing, density-inhibited cultures. The carcinogenic hydrocarbon 9,10-dimethyl-1,2-benzanthracene stimulates DNA synthesis and 2-DOG uptake in serum-deprived cultures at those concentrations which also cause morphological changes in the culture. Other carcinogenic hydrocarbons, which produce no morphological changes in the culture do not stimulate DNA synthesis. In contrast to these non-specific effects, DNA synthesis which is inhibited by low concentrations of either ethylene diamine tetraacetate (EDTA) or diethylene triamine pentaacetate (DTPA) is stimulated specifically by Zn⁺⁺. These findings are interpreted to mean that certain metals and carcinogens, like a variety of other agents, interact non-specifically with the plasma membrane to initiate a chain of events leading to DNA synthesis, and that one of these events is the liberation of Zn⁺⁺ for enzyme reactions leading to DNA synthesis.     

Separation of meso and racemic 2,3-butanediol by aqueous liquid chromatography

Fermentation of xylose by Klebsiella pneumoniae (ATCC 8724) producers meso and nonmeso 2,3-butaneodiol. The enzyme Kinetic of 2,3-butanediol stereoisomer formation from acetone is currently under study in our laboratory. Modeling of these kinetics requires resolution of meso and racemic 2,3-butanediol and positive identification of these resolved components. We report their resolution by aqueous liquid chromatography on both an analytical and a preparative scale. The resolved stereoisomer were identified by a combination of gas chromatography, gas chromatography/mass spectroscopy, ¹³C-NMR spectroscopy, optical activity, and, melting points of the m-dinitrobenzoyl eaters of meso and racemic 2,3-butanediol. An aqueous liquid chromatographic technique for resolving and qualifying major components of a butanediol fermentation mixture in 40 min is presented.     

Primary induction of metallothionein by dexamethasone in cultured rat hepatocytes

Dexamethasone or Zn⁺⁺ increase the rate of synthesis of the metal-binding protein metallothionein in hepatocyte cultures. Dexamethasone induction of the capacity to synthesize metallothionein is not blocked by cycloheximide. In contrast, the dexamethasone stimulated increase in Zn⁺⁺ uptake is inhibited by cycloheximide. Like Zn⁺⁺, dexamethasone is a “primary inducer” of metallothionein. The glucocorticoid induction of metallothionein in primary cultures of rat hepatocytes is not mediated through elevation of Zn⁺⁺ uptake.     

Studies on the Nucleases Produced by Microorganisms

The properties of crude phosphodiesterase forming 5′-mononucleotide of Pellicularia H-II were investigated on its metal requirement, pH response for activity and so on. The dialyzate of crude PDase against distilled water became partly inactive, but was recovered with Zn⁺⁺, Mn⁺⁺ and Mg⁺⁺, whereas completely inactivated dialyzate against EDTA was restored specifically with only Zn⁺⁺

The optimum pH of PDase activity was 5.0 and that of ribonuclease 4.0. The crude PDase was partially purified by acetone fractionation and Amberlite IRC-50 (XE-64) or CM-cellulose column chromatography. Two PDase and a RNase activities were recognized.

Pellicularia PDase was found to be of new type according to its Zn⁺⁺ dependency and non-activity towards bis-p-nitrophenyl phosphate.     

Calcium regulation of skeletal myogenesis. I. Cell content critical to myotube formation

Summary Primary cultures of embryonic chick pectoral skeletal muscle were used to study calcium regulation of myoblast fusion to form multinucleated myotubes. Using atomic absorption spectrometry to measure total cellular calcium and the⁴⁵Ca-exchange method to determine free cellular Ca⁺⁺, our data suggest that only the free cellular calcium changes significantly during development under conditions permissive for myotube formation (0.9 mM external Ca⁺⁺). Increases in calcium uptake occurred before and toward the end of the period of fusion with the amount approximating 2 to 4 pmol per cell in mass cultures. If the medium [Ca⁺⁺] is decreased to 0.04 mM, as determined with a calcium electrode, a fusion-block is produced and free cell Ca⁺⁺ decreased 5- to 10-fold. Removal of the fusion-block by increasing medium [Ca⁺⁺] results in a release of the fusion-block and an increase in cellular Ca⁺⁺ to approximately 1 pmol per cell during fusion, and higher thereafter. Cation ionophore A23187 produced transient increases in cellular calcium and stimulated myoblast fusion and the final extent of myotube formation only when added at the onset of culture. Results suggest that transient increased calcium uptake alone is insufficient for fusion because critical cellular content in conjunction with permissive amounts of medium [Ca⁺⁺] must exist. The latter suggests further that cell surface Ca⁺⁺ was also critical.     

Lignin and extractives derived inhibitors in the 2,3-butanediol fermentation of mannose-rich prehydrolysates

Summary Mannose, the predominant sugar in southern pine water prehydrolysates, has been fermented to 2,3-butanediol by Klebsiella pneumoniae AU-1-d3. Lignin derivatives and extractives soluble in the water prehydrolysates, however, hindered the butanediol fermentation. Treatments with sequential lime-sulfuric acid or mixed bed ion resins facilitated the butanediol fermentation of the water prehydrolysates. Fermentation inhibitors derived from southern pine lignin and extractives were identified.     

Effect of zinc sulphate on the growth of mono- and multispecies cultures of some marine plankton algae

The effect of zinc sulphate has been investigated in mono- and multispecies cultures of the dinoflagellatesScrippsiella faeroense, Prorocentrum micans andGymnodinium splendens and of the diatomsSchroederella schroederi andThalassiosira rotula. Multiplication rate, in vivo chlorophyll fluorescence, maximum cell densities and Zn-conditioned disturbance of the species equilibrium of the multispecies cultures were used as criteria of sublethal toxic inhibition. In monocultures, the first effect became manifest after addition of 0.01 to 0.1 mg Zn⁺⁺ · l^–1. Diatoms proved to be more sensitive than dinoflagellates. In multispecies cultures, the growth of each species depended on the ratio of the inocula. Interrelation between interspecific competition and Zn-caused decrease in the number of algal cells regulated further growth of the cultures. Algal sensitivity to zinc increased with the number of species combined in the test medium: in a 5-species culture sublethal changes appeared already after addition of 0.005 to 0.01 mg Zn⁺⁺ · l^–1. In a few cases, interspecific competition depressed the growth of some species to an appreciable extent, even in the control cultures. At the resulting low cell numbers, the effect of zinc became apparent only in higher concentrations from 5 to 10 mg Zn⁺⁺ · l^–1. Morphological aberrations became manifest inScrippsiella faeroense and in the diatoms in concentrations from 1 and 0.01 mg Zn⁺⁺ · l^–1 respectively. The results show that multispecies experiments are a more sensitive test method for investigating the influence of zinc on plankton algae than are monoculture experiments. In natural plankton communities, however, the toxicity of heavy metals may become effective at considerably lower limit concentrations; this is suggested by the simplified model investigations in the laboratory.     

Chelation of divalent cations by lomofungin: role in inhibition of nucleic acid synthesis

Lomofungin inhibition of yeast growth and RNA synthesis is prevented by Cu⁺⁺ or Zn⁺⁺ ions which chelate with the antibiotic and prevent its uptake by the cells. EDTA potentiates the inhibition. Mg⁺⁺ ions do not protect $\text{in vivo}$ or against the inhibition of purified bacterial RNA and DNA polymerases. Lomofungin prevents formation of the RNA polymerase. DNA initiation complex, probably by chelation with the firmly bound Zn⁺⁺ of the enzyme.     

Fermentation of glycerol to 1,3-propanediol and 2,3-butanediol by Klebsiella pneumoniae

Klebsiella pneumoniae was shown to convert glycerol to 1,3-propanediol, 2,3-butanediol and ethanol under conditions of uncontrolled pH. Formation of 2,3-butanediol starts with some hours' delay and is accompanied by a reuse of the acetate that was formed in the first period. The fermentation was demonstrated in the type strain of K. pneumoniae, but growth was better with the more acid-tolerant strain GT1, which was isolated from nature. In continuous cultures in which the pH was lowered stepwise from 7.3 to 5.4, 2,3-butanediol formation started at pH 6.6 and reached a maximum yield at pH 5.5, whereas formation of acetate and ethanol declined in this pH range. 2,3-Butanediol and acetoin were also found among the products in chemostat cultures grown at pH 7 under conditions of glycerol excess but only with low yields. At any of the pH values tested, excess glycerol in the culture enhanced the butanediol yield. Both effects are seen as a consequence of product inhibition, the undissociated acid being a stronger trigger than the less toxic diols and acid anions. The possibilities for using the fermentation type described to produce 1,3-propanediol and 2,3-butanediol almost without by-products are discussed. Received: 4 February 1998 / Received revision: 30 March 1998 / Accepted: 13 April 1998     

The combined enzymatic hydrolysis and fermentation of hemicellulose to 2,3-butanediol

Summary Hemicellulose-rich fractions from several agricultural residues were converted to 2,3-butanediol by a combined enzymatic hydrolysis and fermentation process. Culture filtrates from Trichoderma harzianum E58 were used to hydrolyze the substrates while Klebsiella pneumoniae fermented the liberated sugars to 2,3-butanediol. Approximately 50–60% of a 5% (w/v) xylan preparation could be hydrolyzed and quantitatively converted to 2,3-butanediol using this procedure. Although enzymatic hydrolysis was optimal at pH 5.0 and 50° C, the combined hydrolysis and fermentation was most efficient at pH 6.5 and 30° C. Combined hydrolysis and fermentation resulted in butanediol levels that were 20–40% higher than could be obtained with a separate hydrolysis and fermentation process. The hemicellulose-rich water-soluble fractions obtained from a variety of steam-exploded agricultural residues could be readily used by the combined hydrolysis and fermentation approach resulting in butanediol yields of 0.4–0.5 g/g of reducing sugar utilized.     

Study of amino and sulfhydryl sites in the sodium pathway in dog red blood cell membranes

Summary Amino reactive TNBS (2,4,6-trinitrobenzene sulfonic acid), SITS (4-acetamido-4-isothiocyano-stilbene-2-2-disulfonic acid), and Zn⁺⁺, and SH reactive Hg⁺⁺ were employed to study sodium channels in dog red blood cells. Simultaneous modification of the membrane with both a SH and an amino modifier results in an increase in Na⁺ permeability which is equal to the sum of their individual effects. This indicates that SH and amino sites are separate units. Three lines of evidence indicate that the amino sites are more superficial than the SH sites. (1) Pretreatment with an amino modifier decreases the effectiveness of subsequent SH modification. (2) SITS, a nonpenetrating amino reagent, enhances Na⁺ permeability while DTNB, a nonpenetrating SH modifier, is ineffective. (3) Pretreatment of amino sites decreases the apparent affinity of Hg⁺⁺ for SH sites. In addition, three lines of evidence indicate that TNBS and Zn⁺⁺ modify different amino sites. First, simultaneous modification with TNBS and Zn⁺⁺ results in an increase in Na⁺ permeability equal to the sum of their individual effects. Secondly, Zn⁺⁺ causes an increase in Na⁺ permeability in cells previously treated with TNBS. Finally, the pH dependence of Zn⁺⁺ modification is opposite that for TNBS modification. These pH experiments suggest that Zn⁺⁺ enhances Na⁺ permeability by reacting with unprotonated amino sites while TNBS modifies protonated amino sites. It is concluded that the sodium permeability of dog red blood cells is normally limited by superficial amino sites and deeper sulfhydryl sites in the sodium channels.     

Electrical stability of erythrocytes in the presence of divalent cations

Erythrocytes suspended in a medium of low ionic strength lyse under the effect of an exponential electrical pulse. The percentage of haemolysed cells decreases several-fold in the presence of divalent cations. The protective action of the ions studied increases in the following order: Ca⁺⁺, Mg⁺⁺, Zn⁺⁺. It is assumed that divalent ions bind to the negative charges of the lipid and protein molecules and reduce their electrostatic repulsion, which results in stabilization of the membranes.