Über eine Bertalanffy-analoge Fluorochromierung mit 3-Dimethylamino-6-methoxyacridin

Zusammenfassung Drei neue Acridinfarbstoffe, 3-Dimethylamino-6-methoxyacridin 1, 3-Amino-6-methoxyacridin 2 und 3-Amino-7-methoxyacridin 3 wurden synthetisiert und ihre Eigenschaften als Fluorochrome für LM- und HeLa-Zellen untersucht. Die Substanzen sind Basen (pK_A: 1 8,76; 2 8,01; 3 7,65), die in neutralem und in saurem wäßrigem Medium Kationen bilden durch Protonierung des Aza-Stickstoffatoms vom Heterocyclus. Die Fluorochrome färben fixierte LM- und HeLa-Zellen bei pH=6. Die Fluoreszenz zeigt Metachromasie in Analogie zur Färbung nach Bertalanffy mit Acridinorange AO. Die Färbung hat höhere Photostabilität als bei AO. Die besten Eigenschaften als Fluorochrom hat 1, das im Detail untersucht wurde.Die Vorschrift der Färbung wurde optimiert. Kerne gefärbter Zellen fluoreszieren gelb-grün, Cytoplasma und Nucleoli orange bis braun-rot. Enzymatische Abbauversuche zeigen, daß der Farbstoff in den Kernen an DNA, im Cytoplasma bzw. den Nucleoli an RNA gebunden ist.Die Absorptions- und Emissionsspektren der fluorochromierten Zellen wurden mikrospektralphotometrisch untersucht. Die Absorptionsspektren von Kern und Cytoplasma sind sehr ähnlich. Das Maximum der längstwelligen Absorptionsbande beobachtet man bei beiden bei 21400 cm^–1 (467 nm) mit einer Schulter bei ca. 20100 cm^–1 (498 nm).Die Fluoreszenzspektren von Kern und Cytoplasma metachromatisch gefärbter Zellen sind verschieden. Das Maximum der Emission des Cytoplasmas bzw. der Nucleoli, 16200 cm^–1 (617 nm), ist langwellig gegenüber dem Fluoreszenzmaximum des Kerns, 18200 cm^–1 (549 nm), verschoben. Diese Verschiebung verursacht den metachromatischen Fluoreszenzeffekt.In Ergänzung wurde die Konzentrationsabhängigkeit der Absorptions- und Fluoreszenzspektren des Kations 1 in wäßriger Lösung, pH=6, im Konzentrationsintervall 6×10^–6–6×10^–4 M untersucht. Lage und Form der Banden hängen nur weinig von der Konzentration ab: Mittelwert des längstwelligen Absorptionsmaximums ca. 21500 cm^–1 (465 nm), der langwelligen Schulter ca. 20300 cm^–1 (493 nm) und des Fluoreszenzmaximums ca. 18300 cm^–1 (547 nm). Mit steigender Konzentration nimmt der molare Extinktionskoeffizient ab. Diese Abnahme und isosbestische Punkte in den Absorptionsspektren weisen auf die Bildung von Dimeren mit steigender Konzentration hin.Die Absorptionsspektren der metachromatisch gefärbten Zellen und des Farbstoffs in Lösung sind sehr ähnlich. Ein sorgfältiger Vergleich der Spektren macht es wahrscheinlich, daß die DNA-gebundenen Farbstoffkationen im Kern monomer, die RNA-gebundenen im Cytoplasma bzw. den Nucleoli dimer sind. Ähnliches wurde bei AO gefunden. Die spektralen Effekte sind jedoch bei 1 viel kleiner.Die Fluoreszenz der RNA-gebundenen Farbstoffkationen des Cytoplasmas ist stark langwellig gegenüber der Kernfluoreszenz verschoben. Absorptions- und Emissionsspektren lassen sich unter der Annahme deuten, daß der RNA-gebundene Farbstoff des metachromatisch gefärbten Cytoplasmas bzw. der Nucleoli im Grundzustand Dimere D, im ersten angeregten Zustand Excimere E bildet. Die Excimeren sind gegenüber den Dimeren stabilisiert; der Bindungsabstand R zwischen den Molekülen ist verkürzt. Das Potentialschema V(R) für Grund- und ersten angeregten Zustand wird im Detail diskutiert. Danach kann D nur in Absorption, E nur in Emission beobachtet werden. Unsere experimentellen Befunde stehen mit einer Excimerenhypothese in Übereinstimmung.Absorptions-, Fluoreszenz- und Absorptionspolarisationsspektren von 1 (Kation und freie Base) wurden in glasartig erstarrtem Ethanol bei 77 K gemessen. Die Spektren wurden mit quantenmechanischen Modellrechnungen in SCF-CI-PPP-Approximation verglichen. Danach sind die beiden Absorptionsbanden des freien Kations in wäßriger Lösung bei ca. 20300 und 21500 cm^–1 und des gebundenen Kations bei 20100 und 21400 cm^–1 dem 0-0- und 0-1-Übergang der längstwelligen Elektronenbande von 1 zuzuordnen.

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Bertalanffy-analogical fluorescence staining with 3-dimethylamine-6-methoxyacridine

Summary Three new acridine dyes, 3-dimethylamino-6-methyoxyacridine 1, 3-amino-6-methoxyacridine 2 and 3-amino-7-methoxyacridine 3, have been prepared and tested as fluorochromes of LM- and HeLa-cells. The dyes are basic compounds (pK_A: 1 8,76; 2 8,01; 3 7,65) and form cations in neutral or acidic aqueous solutions by addition of a proton to the aza-nitrogen atom of the heterocycle. The fluorochromes stain fixed LM- and HeLa-cells at pH=6. The fluorescence shows metachromasy similar to the staining with acridine orange AO according to the technique of Bertalanffy. But there is less fading of the fluorescence. The dye 1 is the most suitable fluorochrome of the series. It was studied in detail.Using optimized staining conditions the fluorescence of the nucleus is yellow-green that of the cytoplasm and the nucleoli orange or brownish-red. Enzymatic digestion experiments show that the dye cations are bound to DNA in the nucleus and to RNA in the cytoplasm or nucleoli.The absorption and emission spectra of the stained cells have been studied by means of microspectrophotometry. The absorption spectra of the nucleus and the cytoplasm are very similar. The maximum of the long wave length absorption of both occurs at 21400 cm^–1 (467 nm) with a shoulder at ca 20100 cm^–1 (498 nm).The fluorescence spectra of nucleus and cytoplasm of metachromatically stained cells are different. The emission maximum of the cytoplasm and nucleoli, 16200 cm^–1 (617 nm), is red-shifted relative to the maximum of the nucleus, 18200 cm^–1 (549 nm). This shift causes the metachromatic fluorescence effect.In addition we studied the concentration dependence of the absorption and fluorescence spectra of the cation 1 in aqueous solution, pH=6, in the concentration range 6×10^–6–6×10^–4 M. Shape and maximum of the long wave length absorption and emission depend only slightly on the concentration: Mean value of absorption maximum ca 21500 cm^–1 (465 nm), shoulder at ca 20300 cm^–1 (493 nm), fluorescence maximum ca 18300 cm^–1 (547 nm). With growing concentration diminishes the molar absorptivity. This decrease in absorptivity and isosbestic points in the absorption spectra indicate the formation of dimers with growing dye concentration.The absorption spectra of the metachromatically stained cells and of the dye in aqueous solution are very similar. A careful comparison of the spectra make it probable that the dye cations bound to DNA in the nucleus are monomer and to RNA in the cytoplasm or nucleoli are dimer. The same has been observed with AO. But our spectral changes are smaller.The fluorescence of the dye cations bound to RNA in the cytoplasm is strongly red-shifted compared to the fluorescence of the nucleus. Absorption and emission spectra of metachromatically stained cytoplasm can be explained on the assumption that the RNA bound dye forms dimers D in the ground state and excimers E in the first excited state. Compared with D the excimers are stabilized and the bond distance R between the molecules is shortened. The potential energy curves V(R) of the ground state and the first excited state are discussed in detail. Accordingly D can only be observed in absorption and E in fluorescence. Our experimental results agree with the excimer hypothesis.Absorption, fluorescence and absorption polarisation spectra of 1 (cation and free base) have been measured in rigid ethanol at 77 K. The spectra are compared with quantum mechanical calculations in SCF-CI-PPP-approximation. According to that the two absorption bands of the free cation in aqueous solution at ca 20300 and 21500 cm^–1 and of the bound cations at ca 20100 and 21400 cm^–1 are classified as 0-0- and 0-1-transitions of the long wave length absorption of 1.

     

Production of acetate by mutant strains of Clostridium thermoaceticum

Summary Mutant strains were derived from Clostridium thermoaceticum ATCC 39 289 by treatment with chemical mutagenic agents and selective enrichment procedures. Some mutant strains exhibited growth when cultured in media containing 20 mabetm (1.75 g l^–1) pyruvate of high-magnesium lime (dolime) above pH 6.0. One strain (G-20) grew and produced acetate when 80 mabetm (7 gl^–1) pyruvate or 50 mabetm (2.3 g l^–1) formate at pH 5.6 was the sole energy source. In a fed-batch process controlled at pH 6.2, this mutant produced 52.5 g l^–1 acetate (equivalent to 72.5 g l^–1 Na acetate) and 67 g l^–1 calcium-magnesium acetate (CMA) in 140 h when dolime was the neutralizing agent, with 93% substrate utilization. This mutant strain holds promise for CMA production due to its better tolerance of dolime and its ability to synthesize high levels of acetic acid. Offprint requests to: S. R. Parekh     

Photomovement of the red alga Porphyridium cruentum (Ag.) Naegeli

Phototaxis of the unicellular red alga Porphyridium cruentum was studied by staining the slime tracks of individual cells as well as with the aid of a population method. Because of the increased straightness of the movement the mean linear velocity of a unilaterally illuminated population exceeds considerably that of an only photokinetically stimulated one. In white light the phototactic reaction is saturated already at 100 lx. The zero threshold lies at about 1 lx. Spectral sensitivity curves of phototaxis obtained at high photon fluence rates (>=10^–11 mol cm^–2 s^–1) display two main peaks which shift against each other at intermediate irradiances and, finally, form a single maximum in the blue range (443 nm) at low photon fluence rates (10^–12 and 10^–13 mol cm^–2 s^–1). Photon fluence rate-response curves reveal that supraoptimal irradiances decrease the phototactic reaction, especially in the range of the highest sensitivity of the cells. The action spectrum of phototaxis was calculated on the basis of the photon fluence rate-response curves. It shows a maximum at 443 nm and shoulder at 416 nm and between 467 and 477 nm. Wavelengths longer than 540 nm are phototactically inactive even at very high irradiances (25 W m^–2). Thus, this is the first phototactic action spectrum of a biliprotein-containing organism which does not indicate the participation of biliproteins in the absorption of phototactically active light. DCMU and potassium iodide have no specific effects on phototaxis.Abbreviation DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea     

Secondary production of Rhyacophila minora,Ameletus sp., and Isonychia bicolor from streams of low and circumneutral pH in the Appalachian Mountains of West Virginia

We estimated the secondary production of Rhyacophila minora, Ameletus sp., and Isonychia bicolor in three acidic streams and one circumneutral stream in Randolph County, West Virginia. Quantitative benthic samples were collected monthly from these second-order streams from November 1990 to October 1991. Mean pH values in the acidic streams were 4.5, 4.8, and 4.8, and mean pH in the circumneutral stream was 6.7. Production estimates for Rhyacophia minora in the acidic streams were 49.6, 19.2, and 15.8 mg m^–2 y^–1. Production of R. minora in the circumneutral stream was 1.0 mg m^–2 y^–1. Ameletus sp. production estimates for the acidic streams were 144.8, 176.8, and 208.3 mg m^–2 y^–1. Ameletus sp. production in the circumneutral stream was 7.4 mg m^–2 y^–1. Secondary production of I. bicolor in the circumneutral stream was 116.6 mg m^–2 y^–1. No Isonychia were collected from the acidic streams. The higher production of R. minora and Ameletus sp. in the acidic streams may be associated with differences in macroinvertebrate community structure.     

Role of vacuolar adenosine triphosphatase in the regulation of cytosolic pH in hepatocytes

The responses of the cytosolic pH of hepatocytes in suspension to agents affecting the activity of vacuolar adenosine triphosphatase (V-ATPase) and Na/H exchange have been studied. Changes of cytosolic pH were determined both with dual-wavelength excitation (500/440 nm) of the fluorescence of 2,7-bis-(2-carboxyethyl)-5(and 6)-carboxyfluorescein and from the distribution of ¹⁴C-dimethyloxazolidinedione; both methods gave very similar results. Changes of vesicular pH were determined by comparing the fluorescence of fluorescein isothiocyanate-dextran and rhodamine B isothiocyanate-dextran taken up by endocytosis. Nitrate, which inhibits V-ATPase in isolated organelles, induced a concentration-dependent acidification of the cytosol and alkalinization of vesicles, with maximal effects at 25–37.5 mm in each case, indicating that V-ATPase contributes to removal of cytosolic protons. On continued exposure to nitrate, the acidification underwent an amiloride-inhibitable reversal. At the higher concentrations of NO ₃ ^– , both cytosolic acidification and vesicular alkalinization were reduced or absent. Bafilomycin A₁ caused alkalinization of vesicular pH; cytosolic acidification was not observed, possibly because of other ionic exchanges. Recovery of cytosolic pH from an acid load (2 min exposure to 5% CO₂) was sensitive to both 25 mm NO ₃ ^– and to ouabain. The pH dependence of the nitrate effect was tested with media of different pH; the activity was negligible at cytosolic pH 6.2 and rose to a maximum at cytosolic pH 7.3. Treatment of hepatocytes with 0.5–1.0 mm ouabain resulted in an initial alkalinization (0.5–2 min duration) of the cytosol, followed by a spontaneous reversal and, on occasion, further acidification. The alkalinization was blocked by 25 mm NO ₃ ^– , but not by 25 mm gluconate. The results suggest that the cytosolic alkalinization is caused by a stimulation of H⁺ uptake by V-ATPase activity. We conclude that V-ATPases make an important contribution to the regulation of the cytosolic pH of hepatocytes.This work was supported in part by National Institutes of Health B.R.S. Grant 507 RR05417 to Temple University.     

Mass cultivation of the nitrogen-fixing cyanobacteriumGloeotrichia natans,indigenous to rice-fields

Gloeotrichia natans, a nitrogen fixing cyanobacterium common in rice fields in the Philippines, was used for studies to establish key features of its physiology and potential production in outdoor cultures. Under optimal growth conditions (38 °C, pH 8.0, no carbon enrichment) the specific growth rate of rice-field isolate was 0.076 h^–1. The pH of the medium (between 6.5 and 9.0) did not influence the growth rate, but it did affect phycobiliprotein content, as reflected by a change in colour. At pH 7.0 the culture was green-brown, with phycobiliproteins constituting up to 10% of the total protein, while at pH 9.0 the culture was brownish-black and the pigment content was as high as 28% of the total protein. In outdoor cultures the specific growth rate was related directly to cell density in the range of 0.7–1.5 g dry weight 1^–1 at a rate of stirring of 30 rpm, and inversely related to cell density at half this rate. At a stirring of 30 rpm, daily production of outdoor cultures harvested to maintain cell densities of 0.7, 1.15 andw 1.5 g 1^–1 were 14.7, 17.1 and 18.1 g m^–2 d^t, respectively. This rate of production was maintained for more than 45 days. Phycobiliprotein content in the culture kept at a density of 1.5 g 1^–1 reached 14% of the total biomass.     

pH dependent stabilization of S2QA − and S2QB − charge pairs studied by thermoluminescence

The pH dependence of emission peak temperature and decay time of thermoluminescence arising from S₂Q_B ^– and S₂Q_A ^– recombinations demonstrates that a stabilization of S₂Q_B ^– occurs at low pH whereas stabilization of S₂Q_A ^– occurs at high pH. Based on comparative analysis of thermoluminescence parameters of the two types of recombination, we suggest that in the pH range between 5.3 and 7.5, E_m(S₂/S₁) and E_m(Q_A/Q_A ^–) are constant, but E_m(Q_B/Q_B ^–) gradually increases with decreasing pH, while in the pH range between 7.5 and 8.5, an unusual change occurs on S₂Q_A ^– charge pair, which is interpreted as either a decrease in E_m(S₂/S₁) or an increase in E_m(Q_A/Q_A ^–).     

Effects of intensity and quality of light on phycocyanin production by a marine cyanobacterium Synechococcus sp. NKBG 042902

Among 150 strains, including marine cyanobacteria isolated from coastal areas of Japan and a freshwater cyanobacterium from the IAM collection, Spirulina platensis IAM M-135, the marine cyanobacterium Synechococcus sp. NKBG 042902 contained the highest amount of phycocyanin (102 mg/g dry cell weight). We have proposed that the cyanobacterium could be an alternative producer for phycocyanin. The effects of light intensity and light quality on the phycocyanin content in cells of Synechococcus sp. NKBG 042902 were investigated. When the cyanobacterium was cultured under illumination of 25 mol m^–2 s^–1 using a cool-white fluorescent lamp, the phycocyanin content was highest, and the phycocyanin and biomass productivities were 21 mg 1^–1 day^–1 and 100 mg 1^–1 day^–1 respectively. Red light was essential for phycocyanin production by this cyanobacterium. Phycocyanin and biomass production were carried out by the cyanobacterium cultures grown under only red light (peak wavelength at 660 nm) supplied from light-emitting diodes (LED). Maximum phycocyanin and biomass productivities were 24 mg 1^–1 day^–1 and 130 mg 1^–1 day^–1 when the light intensity of the LED was 55 mol m^–2 s^–1.     

Electron transfer in deuterated reaction centers of Rhodobacter sphaeroides at 90 K according to femtosecond spectroscopy data

The primary act of charge separation was studied in P⁺B_A ^– and P⁺H_A ^– states (P, primary electron donor; B_A and H_A, primary and secondary electron acceptor) of native reaction centers (RCs) of Rhodobacter sphaeroides R-26 using femtosecond absorption spectroscopy at low (90 K) and room temperature. Coherent oscillations were studied in the kinetics of the stimulated emission band of P* (935 nm), of absorption band of B_A ^– (1020 nm) and of absorption band of H_A (760 nm). It was found that in native RCs kept in heavy water (D₂O) buffer the isotopic decreasing of basic oscillation frequency 32 cm ^–1 and its overtones takes place by the same factor 1.3 in the 935, 1020, and 760 nm bands in comparison with the samples in ordinary water H₂O. This suggests that the femtosecond oscillations in RC kinetics with 32 cm ^–1 frequency may be caused by rotation of hydrogen-containing groups, in particular the water molecule which may be placed between primary electron donor P_B and primary electron acceptor B_A. This rotation may appear also as high harmonics up to sixth in the stimulated emission of P*. The rotation of the water molecule may modulate electron transfer from P* to B_A. The results allow for tracing of the possible pathway of electron transfer from P* to B_A along a chain consisting of polar atoms according to the Brookhaven Protein Data Bank (1PRC): Mg(P_B)-N-C-N(His M200)-HOH-O = B_A. We assume that the role of 32-cm ^–1 modulation in electron transfer along this chain consists of a fixation of electron density at B_A ^– during a reversible electron transfer, when populations of P* and P⁺B_A ^– states are approximately equal.     

Emulsan quantitation by Nile red quenching fluorescence assay

A Nile red fluorescent technique to quantify 20–200 g ml^–1 of emulsan was developed. Nile red dissolved in DMSO showed an adsorption peak at 552 nm, and emission peak at 636 nm, with molar extinction coefficient of 19,600 cm^–1 M^–1. Nile red fluorescence in DMSO was proportionally quenched by emulsan and the quenching was time-dependent. The assay was used to follow the production of emulsan by cultures of Acinetobacter venetianus RAG-1.     

Modelluntersuchungen zur Struktur des purpurnen Farbstoffkomplexes der Giemsa-Färbung

Summary Nuclei of Giemsa stained cells show a purple coloration, which is generated by a complex of DNA, azure B (AB) and eosin Y (EY). The structure of this complex is unknown. Its absorption spectrum shows a sharp and strong band at 18 100 cm^–1 (552 nm), the so called Romanowsky band (RB). It is possible to produce the complex outside of the cell, but it is cubersome to handle. Easier to handle is a purple complex composed of chondroitin sulfate (CHS), AB and EY, which also shows a sharp and strong RB at 18100 cm^–1 in the absorption spectrum. This CHS-AB-EY complex is a model for the DNA-AB-EY complex of Giemsa stained cell nuclei. We tried to investigate its structure.In the first step of the staining procedure CHS binds AB cations forming a stable CHS-AB complex. In the case of saturation each anionic SO ₄ ^– - and COO^–-binding site of CHS is occupied by one dye cation and the complex has 1:1 composition. It has a strong and broad absorption band with its maximum at ca. 18000 cm^–1 (556 nm). In the second step the CHS-AB complex additionally binds EY dianions forming the purple CHS-AB-EY complex with its RB at 18100 cm^–1. This band can be clearly distinguished from the broad absorption of the bound AB cations. RB is generated by the EY chromophore, whose absorption is shifted to longer wavelength by the interaction with the CHS-AB framework.The CHS chains of the CHS-AB and CHS-AB-EY complexes can be mechanically aligned in a preferred direction k. Fine films of highly orientated complexes were prepared with a special technique and studied with a microspectrophotometer equipped with a polarizer and an analyzer. They are birefringent and dichroic-the more birefringent, the better the mechanical orientation. The sites of best orientation within the film were selected according to the quality of the birefringence. We measured the absorption of these regions with linearly polarized light. By setting the polarizer (e _p parallel () or perpendicular () to k, we found that the transition moment m _AB of the long wave-length absorption of AB in the CHS-AB and the CHS-AB-EY complexes is polarized almost perpendicular to the preferred direction k, m _AB k. But the transition moment m _EY of EY in CHS-AB-EY is polarized parallel to k, m _EY k. The transition moments m _AB and m _EY lay in the molecular plane in the direction of the long axes of the AB and EY chromophores, respectively. Therefore, in both CHS-AB and CHS-AB-EY the long axes of the AB molecules are approximately perpendicular to the CHS chain; but in CHS-AB-EY the long axes of the EY chromophore are parallel to the chain of the biopolymer. This structure is somewhat surprising. In the CHS-AB-EY dye complex the chromophores of AB and EY are not parallel but approximately perpendicular to each other.     

Investigation of electrophysiological responses of Neurospora crassa to blue light

The influence of light in a spectrum range of 350–500 nm 20 W m^-2 (20,000 erg · cm^-2 · s^-1) has been studied in the mycelial cells of Neurospora crassa. Light-induced input resistance and membrane potential changes can be measured by means of intracellular microelectrodes. The value of the input resistance reached maximum after a 2–5 min illumination. The maximum hyperpolarization of the cell membrane reaching 30–40 mV was observed after 20–25 min illumination, when the input resistance values did not differ significantly in the illuminated and non-illuminated cells.     

Seasonal changes in composition and biomass of epilithic algal floras of a chalk stream and a soft water stream with estimates of production

Microscopic epilithic algae in the River Itchen at Otterbourne near Southampton and in the Ober Water in the New Forest were studied during 1984 and 1985. The River Itchen rises from chalk springs and has a steady pH near 8.2 and a mean alkalinity of 236 mg HCO₃ 1^–1; at the study site the river is about 16 m wide and 20 cm deep, with a mean flow rate of 0.33 m s^–1 and a discharge ranging through the year between 0.34 and 2.46 m³ s^–1. The Ober Water, which drains sands and gravels, has a pH between 6.9 and 7.2 and a mean alkalinity of about 50 mg HCO₃ 1^–1; at the study site it is about 6 m wide, with a mean flow rate of 0.27 m s^–1 and a discharge ranging through the year between 0.08 and 1.0 m³ s^–1.Epilithic algae removed from the pebbles that form the major part of the beds of both streams show seasonal changes in abundance and composition. Diatoms peaked in April/May and dominate the epilithic flora in both streams, comprising 70–95% of all algal cells; highest numbers of chlorophytes occurred in summer and cyanophytes increased in autumn. The species composition of the epilithic flora in the two streams was different, as was the population density; algal cell numbers ranged between 500 and 7000 cells mm^–2 of stream floor in the River Itchen and between 8 and 320 cells mm^–2 of stream floor in the Ober Water. The chlorophyll a content of epilithic algae in the River Itchen ranged between 115 and 415 mg m^–2 of stream floor, representing an annual mean biomass of about 8 g m^–2, whereas in the Ober Water a chlorophyll a content of 2.2 to 44 mg m^–2 of stream floor was found, representing an annual mean biomass of about 1 g m^–2. Cautious estimates of the annual production of epilithic algae in these streams suggest a value of about 600 g organic dry weight m^–2 in the River Itchen and about 75 g m^–2 in the Ober Water.     

Physiological adsorption of Sulfolobus acidocaldarius on coal surfaces

Summary The adsorption of Sulfolobus acidocaldarius on bituminous coal surfaces and the respiration rate during adsorption at 70° C were enhanced at pH 1.0–2.0, in comparison with those at pH 3.0–5.0. The maximum number of bacterial cells adsorbed per unit area of coal attained a maximum (1.4 × 10¹¹ cells/m²) at pH 2.0. The rate of desulphurization at pH 2.2–2.5 was higher than at other pHs tested. Micrographs of S. acidocaldarius obtained by TEM and SEM indicated that the cells were adsorbed to the coal surfaces by extracellular slime. Specific inhibitors of membrane-bound ATPase (NaF, 20 mm) and respiration (NaN₃, 1 mm; KCN, 1 mm) had pronounced effects on suppressing adsorption. The amount of S. acidocaldarius adsorbed decreased when the coal particles were leached in advance with 2.0 m HNO₃. These facts lead to the conclusion that the adsorption of S. acidocaldarius on coal surfaces requires physiological activity relatd to respiration or energy conversion. Offprint requests to: V. B. Vitaya     

Biomass and production of Argyrodiaptomus furcatus,a tropical calanoid copepod in Broa Reservoir,southern Brazil

The biomass and the production of Argyrodiaptomus furcatus (Sars), the most abundant copepod in Broa Reservoir (São Carlos, São Paulo State), were estimated, determining in the laboratory the development time and the quantity of organic carbon and establishing the relationship between these two parameters. The daily production was calculated from P = B(1- e^gt) and the annual production was obtained by integrating daily production against time. The maximum production of Argyrodiaptomus furcatus in the reservoir depends on the region considered and on the period of the year. The maximum production was 45.15 mg C m^–3d^–1 in March, 1976 at station II, region of macrophytes and 6.74 mg C m^–3d^–1 at station IV, near the dam. The mean production for the year is 6.26 mg C m^–3d^–1 at station II and 1.43 mg C m^–3d^–1 at station IV.     

Nitrosylation of rabbit ferrous heme-hemopexin

Hemopexin (HPX) serves as a trap for toxic plasma heme, ensuring its complete clearance by transportation to the liver. Moreover, HPX-heme has been postulated to play a key role in the homeostasis of nitric oxide (NO). Here, the thermodynamics for NO binding to rabbit ferrous HPX-heme as well as the EPR and optical absorption spectroscopic properties of rabbit ferrous nitrosylated HPX-heme (HPX-heme-NO) are reported. The value of the dissociation equilibrium constant for NO binding to rabbit ferrous HPX-heme (i.e., H) is (1.4±0.2)×10^–7 M, at pH 7.0 and 10.0 °C; the value of H is unaffected by sodium chloride. At pH 7.0, rabbit ferrous HPX-heme-NO is a six-coordinate heme-iron species, characterized by an X-band EPR spectrum with an axial geometry and by =146 mM^–1 cm^–1 at 419 nm. At pH 4.0, rabbit ferrous HPX-heme-NO is a five-coordinate heme-iron species, characterized by an X-band EPR spectrum with three-line splitting centered at 334 mT and by =74 mM^–1 cm^–1 at 387 nm. The pK_a value of the reversible pH-induced six- to five-coordinate spectroscopic transition is 4.8±0.1 in the absence of sodium chloride and 4.3±0.1 in the presence of 1.5×10^–1 M sodium chloride. This result is in agreement with the effect of sodium chloride on rabbit HPX-heme stability. The present data have been analyzed in parallel with those of a related heme model compound and heme-protein systems.     

Chondrus crispus (Gigartinaceae,Rhodophyta) tank cultivation: optimizing carbon input by a fixed pH and use of a salt water well

The injection of exogenous carbon into intensively cultivated algal tanks is necessary to insure a maximum growth rate by stabilizing the dissolved inorganic carbon (DIC) pool, but represents the major part of the cultivation cost (ca. 73%). This study was conducted in paddle-wheel tanks ranging in size from 260 m² to 1000 m². Additional carbon was provided by carbon dioxide mixed into the incoming sea water through a tubular reactor. Production vs pH was analysed on 120 growth measurements covering two years of continuous cultivation. Whereas production peaked at pH 8.0–8.2, the economic optimum for pH regulation was in the range 8.4–8.5, where CO₂ injection was greatly reduced (–29%) for only a slight decrease in production (–4%). Expressed as a function of pH level, the specific carbon injection (g c gdw^–1 of Chondrus produced) showed an inverse exponential relationship, whereas gross photoconversion ratio (gdw mol photons^–1) varied according to a second degree equation with a low amplitude. The photoconversion ratio was not improved when the culture was maintained at a DIC concentration higher than the natural equilibrium (0.64 ± 0.11 gdw mol photons^–1 at 2.35 mM and 0.65 ± 0.15 gdw mol photons^–1 at 3.19 MM).A complementary source of carbon was found in underground salt water with a high and stable DIC concentration (10.15 ± 0.25 mmole Cl^–1). The mixing of the well water with natural sea water allowed another economy of CO₂ (–20% at pH 8.5) and nutrients (–12%), the total unitary cost of production being cut by about 17%.     

Spectroscopic evidence for the presence of an iron-sulfur center similar to Fx of Photosystem I inHeliobacillus mobilis

Treatment of membranes ofHeliobacillus mobilis with high concentrations of the chaotropic agent urea resulted in the removal of the iron-sulfur centers F_A and F_B from the reaction center, as indicated by EPR spectra under strongly reducing conditions. In urea-treated membranes, transient absorption measurements upon a laser flash indicated a recombination between the photo-oxidized primary donor P798⁺ and a reduced acceptor with a time constant of 20 ms at room temperature. Benzylviologen, vitamin K-3 and methylene blue were found to accept electrons from the reduced acceptor efficiently. A differential extinction coefficient of 225–240 mM^–1 cm^–1 at 798 nm was determined from experiments in the presence of methylene blue. Transient absorption difference spectra between 400 and 500 nm in the presence and absence of artificial acceptors indicated that the electron acceptor involved in the 20 ms recombination has an absorption spectrum similar to that of an iron-sulfur center. This iron-sulfur center was assigned to be analogous to F_X of Photosystem I. Our results provide evidence in support of the presence of F_X in heliobacteria, which was proposed on the basis of the reaction center polypeptide sequence (Liebl et al. (1993) Proc. Natl. Acad. Sci. USA 90: 7124–7128). Implications for the electron transfer pathway in the reaction center of heliobacteria are discussed.     

Red Drop in the Quantum Yield of Fluorescence of Sonicated Algae

The change of the quantum yield of fluorescence, Φ, with the frequency of exciting light, was investigated in Chlorella, Anacystis, and Porphyridium suspensions, and in sonicates from these cells prepared under aerobic and anaerobic conditions. In case of Chlorella, sonicates were made in acid and in alkaline media (pH 4.65 and 7.80). In the alkaline medium, a drop of Φ towards the longer waves was found to begin at 1.466 × 10⁴ cm^-1 (682 nm) in sonicates, and in suspension. In the acid medium, the drop began at 1.471 × 10⁴ cm^-1 (680 nm), 1.418 × 10⁴ cm^-1 (705 nm), and 1.389 × 10⁴ cm^-1 (720 nm) in suspension, anaerobic sonicate, and aerobic sonicate, respectively. The results indicate that the cause of the change in the red drop is preferential destruction of a long-wave component of chlorophyll a (such as Chl a 693). The amount of this component remaining after sonication is larger in alkaline than in acid sonicates. With Anacystis and Porphyridium, only alkaline suspensions (pH 7.80) could be used for sonication, because in acid medium, the phycobilin-chlorophyll complex is rapidly broken and phycobilin extracted from the cell. In Anacystis, the red drop begins at 1.562 × 10⁴ cm^-1 (640 nm) and 1.538 × 10⁴ cm^-1 (650 nm) in suspension and sonicate, respectively; in Porphyridium, it starts at 1.550 × 10⁴ cm^-1 (645 nm) in both cases. These results suggest that sonication in alkaline medium (pH 7.80) destroys some Chl a 693 in Anacystis, but not in Porphyridium.     

Butyric acid bacteria of the genus <Emphasis Type="Italic">Clostridium</Emphasis> in the bottom sediments of inland basins of different types

The cell numbers and ecological characteristics of the distribution of certain species of butyric acid bacteria (BABs) of the genus Clostridium in the bottom sediments of inland basins of different types were studied using the optimal nutrient media. The seasonal dynamics of clostridial vegetative cells and spores in sediments with different ecological conditions were revealed. The cell numbers of the dominant BAB species were shown to depend on the redox potential of the sediments, the amount and composition of C_org, and the trophic state of the basin in general. C. pasteurianum was found to predominate in eutrophic lakes and reservoirs (5–11 × 10⁶ cells/cm³), C. butyricum and C. felsineum predominated in mesotrophic ones (2–11 × 10⁶ cells/cm³), and C. acetobutylicum was predominant in acidic chthonioeutrophic lakes and reservoirs (0.1–0.5 × 10⁶ cells/cm³). The lowest cell numbers of BABs were found in river sediments, whereas the highest numbers were recorded in the sediments of polysaprobic zones (0.1–1.0 × 10³ and 0.5–2.0 × 10⁷ cells/cm³ respectively).Translated from Mikrobiologiya, Vol. 74, No. 1, 2005, pp. 119–125.Original Russian Text Copyright © 2005 by Dzyuban.