Methodische Studien zur Zytochemie der Leukozytenphosphatasen

Zusammenfassung Die Technik des Nachweises der a. L.-Ph. in Blutausstrichen wird besprochen. Zur Fixierung wird die Methode vonKaplow empfohlen: 90% Methanol, 10% Formol 1:10, 30 sec bei 0°C. Vergleichswerte mit anderen Fixantien werden aufgeführt.Metallionen aktivieren die a. L.-Ph. in derart fixierten Ausstrichen in der Reihenfolge abnehmender Wirksamkeit:Mg⁺⁺-Fe⁺⁺⁺-Co⁺⁺-Mn⁺⁺-Cu⁺⁺-Fe⁺⁺. Die Wirkung aller Ionen erwies sich als stark konzentrationsabhängig. Ni⁺⁺, Zn⁺⁺ und Pb⁺⁺ hemmten ebenfalls konzentrationsabhängig.In den Blutausstrichen ist die Spaltungsgeschwindigkeit des sauren Na--Naphthylphosphats (Azo-Kupplung) weit höher als die des -Glycerophosphats bei maximaler Mg⁺⁺-Aktivierung mit der Calcium-Kobalt-Methode nachGomori-Takamatsu. Die Spaltungsgeschwindigkeit von -Glycerophosphat kann durch Zusetzen kleiner Mengen Fe⁺⁺⁺ und Cu⁺⁺ über die Mg⁺⁺-Aktivierung hinaus gesteigert werden bei Beschleunigung der Anfangsgeschwindigkeit der Hydrolyse.

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Summary A technic is described for demonstrating the activity of alkaline phosphatase in human leucocytes. For fixation the method ofKaplow is recommended: 90 per cent methanol +10 per cent formalin 1:10 for 30 seconds at 0°C. Other fixatives are evaluated.Under the conditions of the experiment metal ions activated the alkaline phosphatase of leucocytes in the following order: Mg⁺⁺>Fe⁺⁺⁺>Co⁺⁺>Mn⁺⁺>Cu⁺⁺>Fe⁺⁺.The effectiveness of all these metal ions was to a high degree dependent on concentration. Ni⁺⁺, Zn⁺⁺, and Pb⁺⁺ showed an inhibitory effect also dependent on concentration.In fixed blood smears the velocity of hydrolysis of sodium--naphthylphosphate (simultaneous azo-coupling technic) is far greater than that of Na--glycerophosphate (Ca-Co-method of Gomori-Takamatsu). Adding small amounts of Fe⁺⁺⁺ and Cu⁺⁺ to the incubation medium, it is possible to increase the velocity of hydrolysis of glycerophosphate beyond the values of maximal Mg⁺⁺ activation and simultaneously enhancing the initial velocity of the reaction.

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Ferrous iron dependent nitric oxide production in nitrate reducing cultures of Escherichia coli

l-Lactate-driven ferric and nitrate reduction was studied in Escherichia coli E4. Ferric iron reduction activity in E. coli E4 was found to be constitutive. Contrary to nitrate, ferric iron could not be used as electron acceptor for growth. Ferric iron reductase activity of 9 nmol Fe²⁺ mg^-1 protein min^-1 could not be inhibited by inhibitors for the respiratory chain, like Rotenone, quinacrine, Actinomycin A, or potassium cyanide. Active cells and l-lactate-driven nitrate respiration in E. coli E4 leading to the production of nitrite, was reduced to about 20% of its maximum activity with 5 mM ferric iron, or to about 50% in presence of 5 mM ferrous iron. The inhibition was caused by nitric oxide formed by a purely chemical reduction of nitrite by ferrous iron. Nitric oxide was further chemically reduced by ferrous iron to nitrous oxide. With electron paramagnetic resonance spectroscopy, the presence of a free [Fe²⁺-NO] complex was shown. In presence of ferrous or ferric iron and l-lactate, nitrate was anaerobically converted to nitric oxide and nitrous oxide by the combined action of E. coli E4 and chemical reduction reactions (chemodenitrification).     

Voltammetric studies of Zn and Fe complexes of EDTA: Evidence for the push mechanism

The `push' hypothesis for the antioxidant action of Zn²⁺ is based on its displacement of iron from a low molecular weight pro-oxidant complex. In this study, the chemical plausibility of that proposed function is investigated by cyclic voltammetry. As a model for a pro-oxidative low molecular weight iron complex the Fe^II/IIIEDTA couple was examined. This complex was selected for its well-defined electrochemical, iron stability constants, and similarity to other low molecular weight chelates in physiological fluids in terms of logical binding sites, i.e. amino, and carboxylate groups. Also investigated were iron complexes of nitrilotriacetic acid and DL-glutamic acid. Results demonstrate that approximately 90% of the cyclic voltammetric peak current for Fe^IIIEDTA reduction and the EC′ current for the mediated reduction of H₂O₂ by Fe^II/IIIEDTA (Fenton Reaction) are lost when Zn²⁺ is introduced to a 1:1 molar ratio relative to iron. All experiments were conducted in HEPES buffered solutions at pH 7.4. Iron (II/III) complexes of nitrilotriacetic acid and DL-glutamic acid followed the same trends. Cyclic voltammetric experiments indicate that Zn²⁺ displaces Fe^III from EDTA despite the much larger stability constant for the iron complex (10^25.1) versus zinc (10^16.50). The hydrolysis aided displacement of Fe^III from EDTA by Zn²⁺ is considered by the equilibria modeling program, HySS. With Fe^III hydrolysis products included, Zn²⁺ is able to achieve 90% displacement of iron from EDTA, a result consistent with cyclic voltammetric observations. Published online December 2004     

Dynamic equilibria in iron uptake and release by ferritin

The function of ferritins is to store and release ferrous iron. During oxidative iron uptake, ferritin tends to lower Fe²⁺ concentration, thus competing with Fenton reactions and limiting hydroxy radical generation. When ferritin functions as a releasing iron agent, the oxidative damage is stimulated. The antioxidant versus pro-oxidant functions of ferritin are studied here in the presence of Fe²⁺, oxygen and reducing agents. The Fe²⁺-dependent radical damage is measured using supercoiled DNA as a target molecule. The relaxation of supercoiled DNA is quantitatively correlated to the concentration of exogenous Fe²⁺, providing an indirect assay for free Fe²⁺. After addition of ferrous iron to ferritin, Fe²⁺ is actively taken up and asymptotically reaches a stable concentration of 1–5 m. Comparable equilibrium concentrations are found with plant or horse spleen ferritins, or their apoferritins. After addition of ascorbate, iron release is observed using ferrozine as an iron scavenger. Rates of iron release are dependent on ascorbate concentration. They are about 10 times larger with pea ferritin than with horse ferritin. In the absence of ferrozine, the reaction of ascorbate with ferritins produces a wave of radical damage; its amplitude increases with increased ascorbate concentrations with plant ferritin; the damage is weaker with horse ferritin and less dependent on ascorbate concentrations.     

Nachweis von saurer Phosphatase in gezüchteten Hühnerherzmyoblasten

Zusammenfassung Ein verbessertes Verfahren zur Durchführung der Gomori-Reaktion gewährleistet die lagegetreue Darstellung der sauren Phosphatase in gezüchteten Hühnerherzmyoblasten. Enzymaktiv sind die nach Verfütterung von eiweißhaltiger Nahrung regelmäßig auftretenden Proteinspeicher, hier Cytosomen genannt, sowie die Cisternen und Vesikel der Golgi-Apparate. Zu Beginn der Interphase weisen die Myoblasten in der Regel bedeutend weniger enzymaktive Cytosomen und Golgi-Apparate auf als in älteren Interphasestadien. Die Golgi-Vesikel trifft man nur in der Golgi-Zone an. Zwischen den Cytosomen und den Golgi-Apparaten besteht eine enge Lagebeziehung. Alle Cytosomen und auch die gelegentlich auftretenden Cytolysomen enthalten vesikuläre Elemente in der Größenordnung der Golgi-Vesikel. Aus diesen Befunden wird geschlossen, daß die Golgi-Cisternen saure Phosphatase produzieren oder aktivieren und durch Abschnürung von Vesikeln an die Cytosomen übermitteln, die nunmehr Lysosomen-Charakter annehmen.

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Summary An improved method of the Gomori-technique enables the exact localization of acid phosphatase activity within cultured chicken heart myoblasts. Enzym activity is found in the protein storing granules, the so-called cytosomen, which usually appear within the myoblasts, when albumin has been added to the culture medium, as well as in the cisternae and vesicles of the Golgi apparatus. In early stages of interphase, the cells generally contain much fewer enzym-active cytosomes and Golgi apparatus than in later stages.The Golgi vesicles are only found in the Golgi zone. There is a close, topographical relation between the cytosomes and the Golgi apparatus. All cytosomes as well as the cytolysomes which are occasionally found in the myoblasts, enclose vesicular elements of the size of the Golgi vesicles. From these observations it may be concluded that the Golgi cisternae produce or activate acid phosphatase which is transfered to the cytosomes by the vesicles originating from the Golgi cisternae. Thus the cytosomes assume the character of lysosomes.

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Die Arbeit wurde durch eine Sachbeihilfe der Deutschen Forschungsgemeinschaft ermöglicht. Herrn Prof. Dr. R. Danneel danke ich für beratende Hilfe, Frl. I. Rosocha, Frl. I. Spieckermann und Frl. S. Kliewer für technische Assistenz.     

Outgrowth of sympathetic adrenergic neurons in mice treated with a nerve-growth factor (NGF)

Summary Various tissues from mice treated with a nerve-growth factor (NGF) were studied with the histochemical technique ofFalck andHillarp, which visualizes the adrenergic transmitter in the sympathetic postganglionic neurons. Growth stimulation was detectable in all parts of the sympathetic adrenergic neurons. An increased density of the adrenergic ground plexus was observed in e.g. the iris, submaxillary and parotid glands, blood vessels and intramural ganglionic plexuses of the intestinal tract. Normally non-innervated tissues were also found to contain a considerable number of adrenergic terminals. Of special interest is the striking increase in number of adrenergic terminals in various types of autonomic ganglia, in all probability with an inhibitory effect on ganglionic transmission.This investigation was supported by research grants from the Swedish Medical Research Council (B67-12x-714-02), Magnus Bergwalls stiftelse and Stiftelsen Therese och Johan Anderssons Minne. The skillful technical assistance of MissBarbro Riese is gratefully acknowledged.     

Repetitive somatic embryogenesis in peanut cotyledon cultures by continual exposure to 2,4-d

Somatic embryos from immature cotyledons in peanut (Arachis hypogaea) were initiated on media supplemented with 2,4-dichlorophenoxyacetic acid (2,4-d). Over 90% primary embryogenesis and 41–46% repetitive embryogenesis were obtained 12 weeks after initiation by maintaining embryogenic cultures on medium containing 20 mg 1^-1 2,4-d. Maintenance of cultures on medium with 30 or 40 mg I^-1 2,4-d resulted in lower primary and secondary embryogenesis, and proliferation of nonembryogenic callus. Transfer of embryogenic cultures to a secondary medium with 10 or 20 mg I^-1 2,4-d significantly enhanced secondary embryogenesis compared to basal medium without the growth regulator. The use of Murashige & Skoog versus Finer's media had no significant effect on embryogenesis (85–95%), repetitive embryogenesis (11–37%) or mean embryo number. Secondary embryogenesis was also maintained for over one year by repeated subculture of isolated somatic embryos on medium with 20 mg I^-1 2,4-d.Abbreviations B5 Gamborg et al. medium (Gamborg et al. 1968) - 2,4-d 2,4-dichlorophenoxyacetic acid - FN Finer & Nagasawa medium (Finer & Nagasawa 1968) - MS Murashige & Skoog medium (Murashige & Skoog 1962)     

Fine structural localization of acid phosphomonoesterase in the brush border region of osteoclasts

Summary The fine structural localization of acid phosphomonoesterase was studied in osteoclasts of fracture callus in the rat. In addition to being present in the Golgi apparatus and the lysosomes, reaction product was precipitated over tubular elements, vesicles, and vacuoles located subjacent to the brush border. The findings are compatible with the idea that the calcified matrix can be destroyed by the combined effects of absorption with subsequent intracellular digestions in osteoclasts, and actual secretion of lysosomal enzyme to the extracellular space.This study was supported by grants from the Swedish Medical Research Council (Project no. B72-12X-1006-07A) and Konung Gustav V:s åttioårsfond". The assistance of Miss Silwa Mengarelli and Mrs. Britt-Marie Åkerman is gratefully acknowledged.     

Observations on the localization of arylsulphatase activity in renal cortical tubules

Summary The fine structural localization of arylsulphatase in rat kidney cortex was investigated using p-nitrocatechol sulphate as substrate and lead as capturing ion. The studies included observations of the effects of different modes of fixative application in order to define optimal conditions for the histochemical procedure. Reaction product (lead sulphate) was constantly precipitated in the cytosomes in cells of proximal and distal convoluted tubules and collecting ducts. Previous studies have demonstrated that these same organelles contain acid phosphatase and appear to correspond to lysosomes in biochemically isolated fractions from renal cortex. The observations are compatible with the notion that most of the cytosomes in renal cortical tubules and collecting ducts contain both acid phosphatase and arylsulphatase. The constant absence of precipitate in microbodies of proximal tubule cells supported the assumption that these organelles are functionally different from cytosomes, and may correspond to peroxisomes.     

Evidence for Negative Charge in the Conduction Pathway of the Cardiac Ryanodine Receptor Channel Provided by the Interaction of K+ Channel N-type Inactivation Peptides

We have investigated the interaction of two peptides (ShB — net charge +3 and ShB:E12KD13K — net charge +7) derived from the NH₂-terminal domain of the Shaker K⁺ channel with purified, ryanodine-modified, cardiac Ca²⁺-release channels (RyR). Both peptides produced well resolved blocking events from the cytosolic face of the channel. At a holding potential of +60 mV the relationship between the probability of block and peptide concentration was described by a single-site binding scheme with 50% saturation occurring at 5.92 ± 1.06 μm for ShB and 0.59 ± 0.14 nm for ShB:E12KD13K. The association rates of both peptides varied with concentration (4.0 ± 0.4 sec⁻¹μm ⁻¹ for ShB and 2000 ± 200 sec⁻¹μm ⁻¹ for ShB:E12KD13K); dissociation rates were independent of concentration. The interaction of both peptides was influenced by applied potential with the bulk of the voltage-dependence residing in K_off. The effectiveness of the inactivation peptides as blockers of RyR is enhanced by an increase in net positive charge. As is the case with inactivation and block of K⁺ channels, this is mediated by a large increase in K_on. These observations are consistent with the proposal that the conduction pathway of RyR contains negatively charged sites which will contribute to the ion handling properties of this channel. Received: 15 December 1997/Revised: 13 March 1998     

Characterization of the Na+/H+ Exchanger in the Luminal Membrane of the Distal Nephron

In the rabbit as well as the rat, a Na⁺/H⁺ exchanger is expressed in the apical membrane of both the proximal and distal tubules of the renal cortex. Whereas the isoform derived from the proximal tubule has been extensively studied, little information is available concerning the distal luminal membrane isoform. To better characterize the latter isoform, we purified rabbit proximal and distal tubules, and examined the ethylpropylamiloride (EIPA)-sensitive ²²Na uptake by the luminal membrane vesicles from the two segments. The presence of 100 μm EIPA in the membrane suspension decreased the 15 sec Na⁺ uptake to 75.70 ± 4.70% and 50.30 ± 2.23% of the control values in vesicles from proximal and distal tubules, respectively. The effect of EIPA on 35 mm Na⁺ uptake was concentration dependent, with a IC₅₀ of 700 μm and 75 μm for the proximal and distal luminal membranes. Whereas the proximal tubule membrane isoform was insensitive to cimetidine and clonidine up to a concentration of 2 mm, the 35 mm Na⁺ uptake by the distal membrane was strongly inhibited by cimetidine (IC₅₀ 700 μm) and modestly inhibited by clonidine (IC₅₀ 1.6 mm). The incubation of proximal tubule suspensions with 1 mm (Bu₂) cAMP decreased the 15-sec EIPA-sensitive Na⁺ uptake by the brush border membranes to 24.1 ± 2.38% of the control values. Unexpectedly, the same treatment of distal tubules enhanced this uptake by 46.5 ± 10.3%. Finally, incubation of tubule suspensions with 100 nm phorbol 12-myristate 13-acetate (PMA) decreased the exchanger activity to 58.6 ± 3.04% and 79.7 ± 3.21% of the control values in the proximal and distal luminal membranes, respectively. In conclusion, the high sensitivity of the distal luminal membrane exchanger to various inhibitors, and its stimulation by cAMP-dependent protein kinase A, indicate that this isoform differs from that of the proximal tubule and probably corresponds to isoform 1. Received: 6 March 1998/Revised: 6 July 1998     

Iron deprivation in cyanobacteria

Iron is an essential component of electron transport in almost all living organisms. It is particularly important to phototrophs like cyanobacteria because 22–23 irons are required for a complete functional photosynthetic apparatus. Since the low solubility of Fe⁺⁺⁺ above neutral pH in oxic ecosystems severely limits the biological availability of iron to aquatic microorganisms, cyanobacteria and other microbes have developed a number of responses to cope with iron deficiency. Cyanobacterial responses to iron stress include the synthesis of an efficient, siderophore-based system to scavenge iron and the substitution of ferredoxin with flavodoxin. An additional response in cyanobacteria involves the alteration of the light-harvesting apparatus that includes the appearance of a new, iron-stress-induced, photosystem II, chlorophyll-binding protein. Although cytochromec-553 has a potential non-iron-containing replacement in plastocyanin, a copper-containing protein, iron stress appears to favor the utilization of cytochromec-553 because siderophores also bind copper and form a complex that is excluded from the cell.This paper is intended primarily as a review of molecular and physiological responses of actively growing cyanobacterial cultures to conditions of iron stress, where iron is present but essentially insoluble, and to differentiate these responses from iron starvation, where the amount of iron in the system is not sufficient for cell growth.     

Electron Spin Resonance in ATP and RNA

Cu⁺⁺, Mn⁺⁺, and Fe⁺⁺⁺ account for the electron spin resonances observed in certain samples of ATP and RNA. The copper ion seems more loosely bound to these substances than either iron or manganese. A striking similarity is observed between the manganese spectra in manganese RNA, ATP, and ADP suggesting that the binding sites are similar in the three compounds. The similarity of the e.s.r. spectra of iron ATP and of iron and manganese RNA, except for hyperfine spectrum (hfs) in the latter, suggests that the two ions bind similarly in the two compounds. A detailed interpretation of the spectra is lacking however and these conclusions can only be tentative. When manganese TPP and ATP are heated or pH changed the e.s.r. alters indicating a change in the environment of the ion. The sharp 6 line manganese spectrum in both TPP and ATP at pH 1 suggests an almost “free” ion at this pH in the sense of an almost isotropic average environment.     

Kinetics of iron passage through subcellular compartments of rabbit reticulocytes

Summary The kinetics of the separate processes of Fe₂(III)-transferrin binding to the transferrin receptor, transferrin-receptor internalization, iron dissociation from transferrin, iron passage through the membrane, and iron mobilization into the cytoplasm were studied by pulse-chase experiments using rabbit reticulocytes and⁵⁹Fe,¹²⁵I-labeled rabbit transferrin. The binding of⁵⁹Fe-transferrin to transferrin receptors was rapid with an apparent rate constant of 2×10⁵ m ^–1 sec^–1. The rate of internalization of⁵⁹Fe-transferrin was directly measured at 520±100 molecules of Fe₂(III)-transferrin internalized/sec/cell with 250±43 sec needed to internalize the entire complement of reticulocyte transferrin receptors. Subsequent to Fe₂(III)-transferrin internalization the flux of⁵⁹Fe was followed through three compartments: internalized transferrin, membrane, and cytosol.A process preceding iron dissociation from transferrin and a reaction involving membrane-associated iron required 17±2 sec and 34±5 sec, respectively. Apparent rate constants of 0.0075±0.002 sec^–1 and 0.0343±0.0118 sec^–1 were obtained for iron dissociation from transferrin and iron mobilization into the cytosol, respectively. Iron dissociation from transferrin is the rate-limiting step. An apparent rate constant of 0.0112±0.0025 sec^–1 was obtained for processes involving iron transport through the membrane although at least two reactions are likely to be involved. Based on mechanistic considerations, iron transport through the membrane may be attributed to an iron reduction step followed by a translocation step. These data indicate that the uptake of iron in reticulocytes is a sequential process, with steps after the internalization of Fe₂(III)-transferrin that are distinct from the handling of transferrin.     

Early postmortem changes in the Organ of Corti (guinea pig)

Summary Early post mortem changes in the Organ of Corti are described. 15 minutes after death, when kept at room temperature, 20° C (63° F), an oedematous swelling is observed within the cytoplasm of hair cells and nerve endings, the latter being more severe affected. After 30 minutes post mortem the mitochondria of the hair cells have also become significantly swollen. Three hours post mortem the general character of the hair cells is still recognizable, but most of the nerve endings have been completely destroyed. Acknowledgement. We wish to express our appreciation of the skilful technical assistance of Mrs. B. Flock, Miss A.-M. Lundberg, Miss Sonja Löfvenius, Mr. G. Bornholm and Mr. Rune Ragnefjell.This work was supported by grants from the Swedish Medical Research Council, the National Institute of Health grant (no NB 03956-02), the Therese and Johan Anderssons minne, the Gustav and Tyra Svenssons fund and the King Gustav V Research Fund.     

Studies on polyol metabolism in Aspergillus niger

Summary Sorbitol dehydrogenase has been purified about 26 fold from a strain of Aspergillus niger, growing on sorbitol as the sole source of carbon. An absolute specificity of this enzyme for sorbitol, fructose, NAD and NADH was observed. The K _m for sorbitol and fructose were found to be 9.8x10^-5 M and 6.6x10^-4 M respectively. The enzyme was inhibited by pCMB, NaF and other metal ions studied. The enzyme was slightly activated by Fe⁺⁺⁺.Part of this work was presented at the All India Conference of Microbiologists held at Baroda, 1968/69.     

EDTA activation of H2 photoproduction by Rhodospirillum rubrum

Summary The effect of trace elements (Fe, Ni) and chelating compounds on the activity of hydrogen (H₂) uptake (Hup) hydrogenase, nitrogenase and rate and yield of H₂ photoproduction from l-lactate in photosynthetic cultures of Rhodospirillum rubrum was investigated. Hup activity depended on the availability of Ni²⁺ and was inhibited by EDTA (0.3–0.5 mm ethylenedinitrilotetraacetic acid). Addition of EDTA (0.5 mm) to the culture medium caused a nearly complete inactivation of Hup activity and activation of nitrogenase, which was paralleled by a threefold increase in total H₂ photoproduced from lactate. Hup^– mutants, isolated by transposon Tn5 mutagenesis, produced maximally twofold more H₂ than the wild-type. Experiments with different chelating agents [EDTA, NTA (nitrilotriacetic acid), citrate, isocitrate] and varying concentrations of Fe²⁺ and Fe³⁺ showed that photosynthetic growth and nitrogenase activity of R. rubrum were strongly influenced by the iron supply. It is concluded that EDTA enhanced H₂ photoproduction by (I) inhibition of biosynthesis of Hup hydrogenase and (II) mobilization of iron, thereby activating the biosynthesis of the nitrogenase complex. Correspondence to: M. Kern     

Dégradation du DNA par H2O2 en présence d'ions Cu++, Fe++ et Fe+++

The mechanism of degradation of calf thymus DNA by H₂O₂ in dark and light, and in the presence of either Cu⁺⁺, Fe⁺⁺, or Fe⁺⁺⁺ ions has been investigated by following the decrease of molecular weight M?_w by light scattering. Both in the dark and in light, the rate of degradation decreases in the following order: Cu⁺⁺>Fe⁺⁺>Fe⁺⁺⁺. In order to exploit quantitatively the variation of M?_w with time, we calculated the probability p(t) of rupture in a double stranded polymer as a function of the occurence at random of both breaks of the “first kind” (single hits) and of the “second kind” (double hits), when there are caused by any degrading agent. The value of p(t) can then be related to M?_w(t) for the present case of a randomly polydisperse sample of DNA molecules. In the dark, and in the presence of Cu⁺⁺ ions, a degradation of the first kind (which takes place through the simultaneous or successive splitting of both strands of DNA at the same level) is the only one so far observed. The number of degradation sites of the first kind is equivalent to the number of bound Cu⁺⁺ ions in inner sites of DNA. A model is set up to explain the successive breaks of the two strands of the DNA molecule through the formation of a complex (DNA site–Cu⁺⁺-H₂O₂) which exhibits peroxidative properties. The comparison of the degradation induced under these conditions in a native and a sonicated DNA, shows that the specific sites of attack of ultrasonic waves are not specific sites of H₂O₂ action in the presence of Cu⁺⁺ ions. In the dark and in the presence of Fe⁺⁺ or Fe⁺⁺⁺ ions, breaks of the first kind and second kind are superimposed, but the last are predominant. This is ascribed to the low binding of iron ions in inner sites of DNA under these conditions. A large increase in degradation rate of the second kind occurred in the presence of light (with or without added metallic ions and) is ascribed to the action of the free radicals HO^· (and HO₂^·) which arise from the photolysis of H₂O₂. These results are discussed in relation to those obtained by the action of ionizing radiations on aqueous solutions of DNA.     

Characterization of lipase from an alkalophilic yeast sp.

Summary A lipase producing alkalophilic yeast species was isolated, identified, classified and termed as Candida BG-55, from a sample of nickel catalyst of a vegetable oils industry near Chandigarh, INDIA. The lipase from this microorganism had temperature and pH optima of 40°C and 8.5 respectively and was stable at 45°C for 4 hrs. Enzyme activity was stimulated by Ni⁺⁺ and Ca⁺⁺ ions whereas Fe⁺⁺ and Fe⁺⁺⁺ ions inhibited the activity.     

Microelectrode characterization of the basolateral membrane of rabbit S3 proximal tubule

Summary The purpose of this study was to characterize the basolateral membrane of the S3 segment of the rabbit proximal tubule using conventional and ion-selective microelectrodes. When compared with results from S1 and S2 segments, S3 cells under control conditions have a more negative basolateral membrane potential (V _bl=–69 mV), a higher relative potassium conductance (t _K=0.6), lower intracellular Na⁺ activity (A _Na=18.4mm), and higher intracellular K⁺ activity (A _K=67.8mm). No evidence for a conductive sodium-dependent or sodium-independent HCO ₃ ^– pathway could be demonstrated. The basolateral Na–K pump is inhibited by 10^–4 m ouabain and bath perfusion with a potassium-free (0-K) solution. 0-K perfusion results inA _Na=64.8mm,A _K=18.5mm, andV _bl=–28 mV. Basolateral potassium channels are blocked by barium and by acidification of the bathing medium. The relative K⁺ conductance, as evaluated by increasing bath K⁺ to 17mm, is dependent upon the restingV _bl in both S2 and S3 cells. In summary, the basolateral membrane of S3 cells contains a pump-leak system with similar properties to S1 and S2 proximal tubule cells. The absence of conductive bicarbonate pathways results in a hyperpolarized cell and larger Na⁺ and K⁺ gradients across the cell borders, which will influence the transport properties and intracellular ion activities in this tubule segment.