Cyclosporine A-induced toxicity in two renal cell culture models (LLC-PK1 and MDCK)

Renal damage caused by therapeutic treatment with cyclosporine A has been well documented. Clinical experiences have shown that cyclosporine A nephrotoxicity is determined by interstitial fibrosis with tubular atrophy. However, the exact mechanism by which this drug causes nephrotoxicity has not yet been clarified. This study used an in vitro model in an attempt to identify the cellular mechanisms underlying kidney cyclosporine A damage. We used two cell lines with the characteristics of proximal and distal tubule cells (pig kidney proximal tubular epithelial cell line [LLC-PK1] and Madin–Darby canine kidney cell line [MDCK]. The cell lines were treated with cyclosporine A for 24h. After the treatment, the cells were stained with Trypan Blue to estimate cell viability and processed by histochemical reactions to evaluate their cellular metabolism. Four enzymes (acid phosphatase, alkaline phosphatase, lactate dehydrogenase and succinate dehydrogenase) were considered. The cell viability assay showed that the LLC-PK1 cell line was more sensitive to cyclosporine A than MDCK. Remarkably, the LLC-PK1 cells disappeared with cyclosporine A treatment. As for the hydrolytic enzymes, only acid phosphatases showed an increased positivity in the treated LLC-PK1 cells. Similarly, lactate dehydrogenase showed a different activity histochemically. No statistically significant alterations were observed in the succinate dehydrogenase reaction.The cyclosporine A-treated MDCK cell lines did not show any difference in either their hydrolytic or succinate dehydrogenase enzyme positivity with respect to the control line. In contrast, there was a significant increase in lactate dehydrogenase activity. This study allowed the possible mechanism of cyclosporine A-induced damage in renal tubular cells to be evaluated. The enzymatic changes happened rapidly (during the 24h of treatment), suggesting that this alteration was one of the steps by which cyclosporine A induced toxicity. Moreover, since acid phosphatase is a marker of protein catabolism, the variation in the activity of this enzyme, in the LLC-PK1 line only, showed that cyclosporine can induce alterations leading to cellular toxicity. The modifications in lactate dehydrogenase activity, in both lines, suggested that this drug caused cell stress, inducing the production of lactic acid from glucose in the presence of oxygen. In conclusion, cyclosporine A treatment may force LLC-PK1 and MDCK cells to use anaerobic glycolysis preferentially. Further, these enzyme alterations may represent an epiphenomenon or a consequence of cyclosporine A toxicity.     

Morphological and biochemical characterization of primary culture of rabbit proximal kidney tubule cells grown on collagen-IV coated millicell-cm

Summary The aim of this study was to better characterize rabbit proximal kidney tubule cells cultured on collagen IV-coated porous inserts, as compared to the same cells seeded in standard plastic wells. Total protein contents in confluent monolayers on permeable membranes were about twofold higher than those measured in confluent cultures in plastic wells. Microscopy examinations suggested that such a difference was probably due to a higher cell density and to an impressive development of the apical brush-border membrane. Moreover, measurement of unidirectional transport of p-aminohippuric acid and tetraethylammonium bromide confirmed the high polarization level of cultures on porous inserts. Results of methyl(α-d-[U-¹⁴C]glyco)pyranoside uptake suggested that cell phenotype was probably influenced by culture conditions. Analysis of different markers as a function of time in culture showed decreases of alkaline phosphatase (AP), γ-glutamyltranspeptidase (GGT), and Na⁺-K⁺-ATPase activities as well as increases in LDH, ATP, and glutathione levels, similar to those formerly reported for cells cultured in standard plastic plates. However, comparative data from 6-d-old monolayers have shown that AP, GGT, Na⁺-K⁺-ATPase, glutathione reductase (GRED), and selenium-dependent glutathione peroxidase (Se-GPX) activities were 2.8-, 2.6-, 1.6-, 1.2-, and 2.1-fold, respectively, better preserved on precoated permeable membranes. On the other hand, this paper reports for the first time in the literature that GRED and Se-GPX, two phase II detoxification enzymes, were well maintained in cultures of rabbit proximal kidney tubule cells. Our results show that culturing rabbit proximal kidney tubule cells on collagen IV-coated porous membranes was accompanied by an improvement of both morphological and biochemical properties of the cells.     

Regulation of gluconeogenesis in swine kidney proximal tubule cells

Summary Proximal tubule cells were isolated from swine kidney and cultured for periods of more than 30 days. The cells formed confluent monolayers after plating on a collagen surface and they were passaged more than 5 times on this matrix. The cells maintain several metabolic functions of proximal tubule cells, including gluconeogenesis and the ability to respond to epinephrine and parathyroid hormone. Gluconeogenesis, a principal metabolic pathway in proximal tubule cells, was examined as a function of days in culture. The isolated cells showed a nearly constant rate of gluconeogenesis from ¹⁴C-lactate, ¹⁴C-alkaine and 14C-glycerol with no significant loss of activity for at least 30 days in culture. Likewise, the activities of several cytosolic and membrane associated enzymes including, alkaline phosphatase, -glutamyltransferase, fructose-1,6-bisphosphatase and phosphofructokinase were nearly constant over the same time period.The cells responded to treatment with epinephrine and parathyroid hormone, and the rate of gluconeogenesis from ¹⁴C-lactate doubled in the presence of these hormones. The morphological and biochemical evidence obtained in these studies show that the proximal tubule cells isolated from swine kidney provide an excellent well defined system for studying the hormonal regulation of carbohydrate metabolism in this tissue.Abbreviations PTH Parathyroid Hormone - cAMP cyclic 3,5-adenosine Monophosphate     

Pullulanase,an enzyme of starch catabolism,is associated with the outer membrane of Klebsiella

Late-log phase cells of Klebsiella sp. 5246 could be converted into spheroplasts with a yield of better than 90% by ethylenediamine tetraacetate/lysozyme treatment in osmotically stabilizing media. Membrane fragments obtained after ultrasonication of spheroplasts were separated by centrifugation to sedimentation equilibrium on a sucrose density gradient. A light membrane fraction with a buoyant density of 1.17±0.02g/cm³ was sought and found to contain the enzymes NADH oxidase, succinate dehydrogenase and D-lactate dehydrogenase. A heavy membrane fraction having a buoyant density of 1.23 ±0.01g/cm³ was characterized by phospholipase A₁ activity and lipopolysaccharide content. By analogy to other gram-negative bacteria, the light and the heavy fraction were assigned, respectively, to the cytoplasmic and the outer membrane of Klebsiella sp. 5246.The organism produced pullulanase in a cellbound form during the exponential phase of growth on soluble starch. Pullulanase was localized exclusively on the outer membrane. Pullulanase is the second protein of the outer membrane with defined enzyme function to become known among gram-negative bacteria, the other one being phospholipase A₁.What had been inferred from physiological studies of growth characteristics on various carbon sources can now be proven directly: Pullulanase implicated in the utilization of branched -glucans in Klebsiella is capable of acting on macromolecular substrates in the environment of the cell by virtue of its association with the outer membrane.Non-Standard Abbreviations EDTA ethylenediamine tetraacetate - SDS sodium dodecyl sulphate - OD optical density List of Enzymes EC 3.2.1. 23 -galactosidase or -D-galactoside galactohydrolase - EC 1.1.1.28 D-lactate dehydrogenase or D-lactate: NAD⁺ oxidoreductase - EC 3.2.1.17 lysozyme or mucopeptide N-acetylmuramoylhydrolase - EC 2.4.1.1 maltodextrin phosphorylase or 1,4--D-glucan: orthophosphate -glucosyltransferase - EC 1.6.99.3 NADH oxidase or NADH: (acceptor) oxidoreductase - EC 3.1.1.32 phospholipase A₁ or phosphatide 1-acylhydrolase - EC 3.2.1.41 pullulanase or pullulan 6-glucanohydrolase - EC 1.3.99.1 succinate dehydrogenase or succinate: (acceptor) oxidoreductase     

Glutathione reductase activity in heterocysts and vegetative cells of the cyanobacterium Nostoc muscorum

Glutathione reductase activity was detected and characterized in heterocysts and vegetative cells of the cyanobacterium Nostoc muscorum. The activity of the enzyme varied between 50 and 150 nmol reduced glutathione· min^-1·mg protein^-1, and the apparent K_m for NADPH was 0.125 and 0.200 mM for heterocysts and vegetative cells, respectively. The enzyme was found to be sensitive to Zn⁺² ions, however, preincubation with oxidized glutathione rendered its resistance to Zn⁺² inhibition. Nostoc muscorum filaments were found to contain 0.6–0.7mM glutathione, and it is suggested that glutathione reductase can regenerate reduced glutathione in both cell types. The combined activity of glutathione reductase and isocitrate dehydrogenase in heterocysts was as high as 18 nmol reduced glutathione·min^-1·mg protein^-1. A relatively high superoxide dismutase activity was found in the two cell types; 34.2 and 64.3 enzyme units·min^-1·mg protein^-1 in heterocysts and vegetative cells, respectively.We suggest that glutathione reductase plays a role in the protection mechanism which removes oxygen radicals in the N₂-fixing cyanobacterium Nostoc muscorum.Abbreviations DTNB 5-5-dithiobis-(2-nitrobenzoic acid) - EDTA ethylenediaminetetra-acetic acid - GR glutathione reductase (EC1.6.4.2) - GSH reduced glutathione - GSSG oxidized glutathione - OPT O-phtaldialdehyde - SOD superoxide dismutase (EC 1.15.1.1)     

Stimulated secretion of lysosomal enzymes induced by drugs in transimmortalized proximal tubule mouse kidney cells

We summarize the results of study of the properties of two models of transimmortalized proximal tubule epithelial cells derived from the kidneys of transgenic mice harboring the SV40 large T and little t antigens/L-pyruvate kinase hybrid gene. The two cell lines, reffered to as PKSV-PCT and PKSV-PR cells, maintained for long-term passages the main biochemical and functional properties from the convoluted and terminal parts of the proximal tubule, respectively from which they were derived. In PKSV-PCT cells, gentamicin induced lysosomal alkalinization, decreased the cellular N-acetyl--D-glucuronidase, and stimulated its secretion in a dose-dependent manner. The results indicate that these models of mouse proximal cultured cells could be suitable models for the study of the cellular action of drugs.Abbreviations MDR multidrug resistance - NAG N-acetyl--D-glucuronidase - PGP P-glycoprotein     

Synthesis of symmetric disulfides as potential alternative substrates for trypanothione reductase and glutathione reductase: Part 1

Summary The synthesis of a series of symmetrical disulfides as potential substrates of trypanothione reductase and glutathione reductase was described. The key intermediate in the synthetic approach was the choice of S-^tbutylmercapto-L-cysteine (1). The spermidine ring in the native substrate, trypanothione disulfide (TSST), was replaced with 3-dimethyl-aminopropylamine (DMAPA), while the-Glu moiety was replaced by phenylalanyl or tryptophanyl residues. The same modifications in the-Glu moiety of glutathione disulfide (GSSG) were applied.     

Kinetic and functional characterization of a membrane-bound NAD(P)H dehydrogenase located in the chloroplasts of Pleurochloris meiringensis (Xanthophyceae)

Using isolated chloroplasts or purified thylakoids from photoautotrophically grown cells of the chromophytic alga Pleurochloris meiringensis (Xanthophyceae) we were able to demonstrate a membrane bound NAD(P)H dehydrogenase activity. NAD(P)H oxidation was detectable with menadione, coenzyme Q₀, decylplastoquinone and decylubiquinone as acceptors in an in vitro assay. K _m-values for both pyridine nucleotides were in the molar range (K _m[NADH]=9.8 M, K _m[NADPH]=3.2 M calculated according to Lineweaver-Burk). NADH oxidation was optimal at pH 9 while pH dependence of NADPH oxidation showed a main peak at 9.8 and a smaller optimum at pH 7.5–8. NADH oxidation could be completely inhibited with rotenone, an inhibitor of mitochondrial complex I dehydrogenase, while NADPH oxidation revealed the typical inhibition pattern upon addition of oxidized pyridine nucleotides reported for ferredoxin: NADP⁺ reductase. Partly-denaturing gel electrophoresis followed by NAD(P)H dehydrogenase activity staining showed that NADPH and NADH oxidizing proteins had different electrophoretic mobilities. As revealed by denaturing electrophoresis, the NADH oxidizing enzyme had one main subunit of 22 kDa and two further polypeptides of 29 and 44 kDa, whereas separation of the NADPH depending protein yielded five bands of different molecular weight. Measurement of oxygen consumption due to PS I mediated methylviologen reduction upon complete inhibition of PS II showed that the NAD(P)H dehydrogenase is able to catalyze an input of electrons from NADH to the photosynthetic electron transport chain in case of an oxidized plastoquinone-pool. We suggest ferredoxin: NADP⁺ reductase to be the main NADPH oxidizing activity while a thylakoidal NAD(P)H: plastoquinone oxidoreductase involved in the chlororespiratory pathway in the dark acts mainly as an NADH oxidizing enzyme.Abbreviations Coenzyme Q₀-2,3-dimethoxy-5-methyl-1,4-benzoquinone - FNR ferredoxin: NADP⁺ reductase - MD menadione - MV methylviologen - NDH NAD(P)H dehydrogenase - PQ plastoquinone - PQ₁₀ decylplastoquinone - SDH succinate dehydrogenase - UQ₁₀ decylubiquinone (2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinone)     

Immunocytochemical localization of Δ3, Δ2-enoyl-CoA isomerase and NADPH-dependent-2,4-dienoyl-CoA reductase in rat kidney

Localization of ³, ²-enoyl-CoA isomerase (ECI) and NADPH-dependent-2,4-dienoyl-CoA reductase (DCR) in the rat kidney was investigated by immunocytochemical techniques. The kidneys were perfusion-fixed and embedded in Epon or LR White. For light microscopy, semi-thin sections of Epon-embedded materials were stained by the immunoenzyme technique after the epoxy resin was removed by treatment with sodium ethoxide. For electron microscopy, ultra-thin sections of LR White-embedded materials were stained by the protein A-gold technique. By light microscopy, the S1 segment of the proximal tubule was most heavily stained for ECI and DCR whilst S2 and S3 segments showed intermediate staining. A weak staining reaction was observed in the distal tubule and the medullary collecting tubule. In the cortical collecting tubule, heavily stained cells were present between weakly stained cells. By electron microscopy, gold particles showing the antigenic sites for ECI were confined mainly to the mitochondria, but few particles were observed in the peroxisomes. Gold labeling for DCR was localized both in the mitochondria and the peroxisomes. The labeling intensity of the peroxisomes was much higher than that of the mitochondria. The results suggest that metabolism of unsaturated fatty acids occurs mainly in the mitochondria and the peroxisomes of the proximal tubule in the kidney.     

Specificity of the transport system for tricarboxylic acid cycle intermediates in renal brush borders

Summary Uptake studies employing renal brush border membranes were used to examine the structural specificity of the TCA cycle intermediate transport system. The kinetics of reciprocal inhibition between succinate and citrate revealed these compounds to be transported by a common mechanism. The Michaelis constant for succinate (0.11mm) was significantly lower than that of citrate (0.28mm), indicating that the system has a higher affinity for succinate than for citrate. The specificity of the transport system was determined from the relative inhibitory constants of 40 organic acids on the transport of succinate. The results established that the system is highly specific for 4-carbon, terminal dicarboxylic acids in thetrans-configuration, including the major intermediates of the TCA cycle. The system is comparatively insensitive to monocarboxylates. Substitution of one of several polar, noncharged residues on the -carbon of succinate permitted interaction of the substrate with the transport system, but substitutions on both the and \-carbons did not. The structural specificity of the system is fundamentally different from that of the dicarboxylate and tricarboxylate exchange systems of mitochondria. The role of this transport system in the reabsorption of TCA cycle intermediates from the proximal tubule is discussed.Abbreviations Tris Tris(hydroxymethy)aminomethane - Hepes N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid     

Turbellaria Phagocata sibirica: Some Enzymes of Carbohydrate and Energy Metabolisms

Activities and properties of some enzymes of carbohydrate and energy metabolisms in free-living turbellaria Phagocata sibirica are studied. The enzymes are studied in various subcellular fractions. A high activity of hexokinase is accompanied by high activity of glucose-6-phosphate dehydrogenase (G6PDG). The level of pyruvate kinase activity is sufficient to provide dissimilation of phosphoenolpyruvate with formation of pyruvate. P. sibirica has highly-active lactate dehydrogenase (LDH) and malate dehydrogenase (MDH); a predominance of MDH activity over LDH and a low activity of phosphoenolpyruvate carboxykinase is revealed. NADP-dependent isocitrate dehydrogenase is found, which is activated by Mn²⁺ and Mg²⁺ and inhibited by salts of heavy metals and p-chloromercuribenzoate. Activities and properties of -ketoglutarate dehydrogenase, succinate dehydrogenase (SDH), and fumarate reductase are studied, and it is concluded that in P. sibirica there is the system of succinate oxidation, whereas the system of fumarate reduction into succinate is absent. Mitochondrial and microsomal fractions from P. sibirica had Mg²⁺- and Ca²⁺-dependent adenosine triphosphatases.     

Quantitative measurement of glucose-6-phosphate dehydrogenase in cortical fractions of the rabbit nephron

Summary A quantitative fluorimetric method is described for estimating the activity of glucose-6-phosphate dehydrogenase in isolated fractions of rabbit nephron from the superficial part of the renal cortex: macula densa, proximal convoluted tubule, distal convoluted tubule and glomerulus. The mean activity in the macula densa region was 2.5×10^–18 mol/m³/min, which was about twice the mean activity of the proximal and distal tubular cells and four times that of the glomeruli. As glucose-6-phosphate dehydrogenase is located in the cytoplasm, the average cytoplasmic enzyme activity of the different tubular cells was calculated: macula densa activity was 4.0×10^–18 mol/m³/min whilst proximal tubular cells showed about a third, and distal tubular cells about a quarter of this activity.     

Fine structure of ureteric duct epithelium in the North Atlantic hagfish (Myxine glutinosa L.)

Summary The epithelial cells lining the ureteric duct in the cyclostome, Myxine glutinosa, have a brush border and show specializations of their apical cytoplasm similar to those observed in absorptive proximal tubule cells in higher vertebrate species. These features and the presence of large and numerous cytosomes, presumed to contain lysosomal enzymes, indicate that the ureteric epithelium has taken over some of the functions of the proximal tubule in the atubular kidney of Myxine. Sparsity of basal cytoplasmic processes and mitochondria in the ureteric duct cells appears to correlate with an inability for active, energy-dependent secretory and ion transport functions.This study has been supported by a grant from the Karolinska Institutet Medical School, Stockholm, Sweden (Therese och Johan Anderssons Minne). The author is indebted to Doctor Bertil Swedmark for permission to use the facilities of Kristineberg Marine Biology Station, Fiskebäckskil, Sweden, where hagfish were caught.     

Function of fumarate reductase in methanogenic bacteria (Methanobacterium)

Methanogenic bacteria contain high activities of fumarate reductase. An interesting hypothesis has recently been advanced that this enzyme, in cooperation with a succinate dehydrogenase, functions in a fumarate-succinate cycle for ATP synthesis. This hypothesis was tested by determining whether [2, 3-³H] succinate loses³H when taken up by growing cells.Methanobacterium thermoautotrophicum was grown on H₂ plus CO₂ in the presence of [U-¹⁴C, 2,3-³H] succinate. The double labelled dicarboxylic acid was found to be incorporated into cell material with the loss of only 30% of tritium. Neither was³H released into H₂O in significant amounts. This finding excludes a catabolic oxidation of succinate to fumarate in the growing cells and thus the operation of a fumaratesuccinate cycle. It is shown that the function of fumarate reductase inM. thermoautotrophicum is to provide the cells with succinate for the synthesis of -ketoglutarate, an intermediate in glutamate, arginine and proline synthesis.     

Die Histotopik einiger Oxydoreduktasen im Hoden von Hund und Katze

Zusammenfassung Im Hoden von Hund und Katze werden folgende Enzyme histochemisch nachgewiesen: NADH-Tetrazoliumreduktase (NADH-T-Red), NADPH-Tetrazoliumreduktase (NADPH-T-Red), Cytochromoxydase (Cyt-Ox), Lactat-Dehydrogenase (LDH), Aldolase (ALD), Alkohol-Dehydrogenase (ADH), Glycerin-1-phosphat-Dehydrogenase (GDH), Glucose-6-phosphat-Dehydrogenase (G-6-PDH), Succinat-Dehydrogenase (SDH), NAD-spezifische Isocitrat-Dehydrogenase (NAD-ICDH). Die starke Fermentaktivität der G-6-PDH und der LDH in den Leydig-Zellen beider Spezies, der relativ hohe Gehalt an histochemisch nachweisbarer ADH in den Zwischenzellen der Katze sowie eine deutliche Reaktion auf GDH in den Sertoli-Zellen der Katze werden diskutiert.

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Summary In the testes of dog and cat the distribution pattern of NADH-tetrazolium reductase, NADPH-tetrazolium reductase, cytochrome oxydase, lactate dehydrogenase, aldolase, alcohol dehydrogenase, -glycerophosphate dehydrogenase, glucose-6-phosphate dehydrogenase, succinate dehydrogenase and NAD specific isocitrate dehydrogenase was studied by histochemical means. The strong reaction of G-6-PDH and LDH in the Leydig cells of both species, the relatively high amount of ADH in the interstitial cells of the cat testis and the principal site of -GPDH in the Sertoli cells of the cat are discussed.

     

Nitrate reductase activity in spheroplasts from Rhodobacter capsulatus E1F1 requires a periplasmic protein

Spheroplasts from Rhodobacter capsulatus E1F1 cells grown in nitrate maintained nitrate uptake and nitrate reductase activity only when they were illuminated under anaerobiosis in the presence of the periplasmic fraction and nitrate. The effects on nitrate uptake and nitrate reductase activity of spheroplasts were observed at low concentrations of periplasmic protein (about 50 x ml^-1). Periplasm from nitrate-grown cells was also required for nitrate reductase activity in spheroplasts isolated from ammonia-grown or diazotrophic cells which initially lacked this enzymatic activity. Both the maintenance of nitrate reductase in spheroplasts from nitrate-grown cells and the appearance of the activity in spheroplasts from diazotrophic cells were dependent on de novo protein synthesis. A periplasmic, 45-kDa protein which maintained the activity of nitrate reductase in spheroplasts was partially purified by gel filtration chromatography of periplasm obtained from nitrate-grown cells.Abbreviations NR nitrate reductase - CCCP carbonyl cyanide m-chlorophenylhydrazone - CAM chloramphenicol     

A novel human hydroxysteroid dehydrogenase like 1 gene (HSDL1) is highly expressed in reproductive tissuesa

We report the cloning and characterization of a novel human hydroxysteroid dehydrogenase like gene (HSDL1) located on human chromosome 16q24.2. The HSDL1 cDNA is 3407 base pair in length, encoding a 309 amino acid polypeptide related to human 17-HSD3. Northern blot reveals that the HSDL1 is highly expressed in testis and ovary. In situ hybridization indicates that the expression of HSDL1 is predominantly increased in the prostate cancer tissue compared with the normal prostate tissue, which suggests that the gene expression is important to the arising of prostate cancer.     

The activities of hydrogenase and enoate reductase in two Clostridium species,their interrelationship and dependence on growth conditions

The enzyme activities of Clostridium La 1 and Clostridium kluyveri involved in the stereospecific hydrogenation of ,-unsaturated carbonyl compounds with hydrogen gas were measured. In C. La 1 the specific activities of hydrogenase and enoate reductase depended heavily on the growth phase and the composition of the medium. During growth in batch cultures on 70 mM crotonate the specific activity of hydrogenase increased and then dropped to about 10% of its maximum value, whereas the activity of enoate reductase reached its maximum in cells of the stationary phase. Under certain conditions during growth the activity ratio hydrogenase: enoate reductase changed from 120 to 1. Thus, the rate limiting enzyme for the hydrogenation can be either the hydrogenase or the enoate reductase, depending on the growth conditions of the cells.The specific activities of ferredoxin-NAD reductase and butyryl-CoA dehydrogenase increased 3-4-fold during growth on crotonate. By turbidostatic experiments it was shown that at constant input of high crotonate concentrations (200 mM) the enoate reductase activity was almost completely suppressed; it increased steadily with decreasing crotonate down to an input concentration of 35 mM.Glucose as carbon source led to high hydrogenase and negligible enoate reductase activities. The latter could be induced by changing the carbon source of the medium from glucose to crotonate. Tetracycline inhibited the formation of enoate reductase.A series of other carbon sources was tested. They can be divided into ones which result in high hydrogenase and rather low enoate reductase activities and others which cause the reverse effect.When the Fe²⁺ concentration in crotonate medium was growth limiting, cells with relatively high hydrogenase activity and very low enoate reductase activity in the stationary phase were obtained. At Fe²⁺ concentrations above 3·10^-7 M enoate reductase increased and hydrogenase activity reached its minimum. The ratio of activities changes by a factor of about 200. In a similar way the dependence of enzyme activities on the concentration of sulfate was studied.In batch cultures of Clostridium kluyveri a similar opposite time course of enoate reductase and hydrogenase was found.The possible physiological significance of this behavior is discussed.Non Standard Abbreviations O.D.₅₇₈ Optical density at 578 nm Dedicated to Professor Dr. O. Kandler on the occasion of his 60th birthday     

Analysis of Aldox n alleles isolated from natural populations of Drosophila melanogaster

Aldox null alleles which were isolated from natural populations in Great Britain and North Carolina were analyzed for complementation. No complementation was observed between any combinations of null alleles for aldehyde oxidase (AO) specific activity in late third-instar larvae and newly emerged adults. AO immunologically cross-reacting material (AO-CRM) was quantitated in all homozygous stocks at both developmental stages as well as all allelic combinations in newly emerged adults. When the adult organism contains only Aldox ⁿ alleles, the polypeptides are not immunologically recognizable or may be rapidly degraded. Larvae and adults have different abilities to degrade mutationally altered enzymatically inactive AO polypeptide or synthesize them differentially. This is indicated by easily measurable AO-CRM levels in late third-instar larvae of Aldox ⁿ homozygotes, while newly emerged adult Aldox ⁿ homozygotes have very little, if any, AO-CRM. Newly emerged adult heterozygotes of Aldox ⁿ /Aldox ⁺ do have increased AO-CRM, indicating that the Aldox ⁿ alleles can code for a polypeptide which can be rescued if Aldox ⁺ gene product is present. Heterozygotes containing an Aldox ⁺ allele with a deficiency for the Aldox region produce 74.2% of the AO-CRM found in Aldox ⁺ homozygotes. This may indicate the presence of trans-acting factors which serve to activate gene expression in a system in which each gene copy is not maximally expressed.This work was supported by an Alberta Heritage Foundation for Medical Research Establishment Grant and a Natural Sciences and Engineering Research Council of Canada Operating Grant.     

Expression of multiple cytochromeb 556 species in aerobically grownEscherichia coli

Cytochrome with an-band absorption maximum at 556 nm (77K) in the reduced minus oxidized spectrum is observed in membrnes fromEscherichia coli grown aerobically on most carbon sources. Previous work has suggested that this adsorption peak is due to cytochromeb ₅₅₆ of succinate dehydrogenase and to cytochromeo. We show here, by partial purification of the membrane cytochromes, that at least two other cytochromes with absorption maxima at 556 nm contribute to this peak. One of these cytochromes is associated with growth ondl-lactate. The other is formed under conditions of low aeration and has hydroperoxidase activity.