Studies on the cyanophytes of Cuba 4–6

The article comprises three subsequently published studies of the taxonomy of Cuban cyanophyte/cyanobacterial flora: (4)Lyngbyopsis willei: This oscillatorialean genus and species, described byGardner (1927) from mountain creeks in Puerto Rico, was found more than 50 years later in similar localities in Cuba. The morphological variability of the Cuban populations is described and similarity with the genusSchizothrix (sect.Inactis) discussed. —(5)Cylindrospermum-species: The morphological variability of twenty-oneCylindrospermum-populations collected in Cuba was studied, documented by graphical methods and compared with the published data. Four new taxa were recognized (C. minutissimum v.rinoi, C. zonatum, C. bourrellyi, andC. desikacharyi). The other populations belong to the variation ranges ofC. breve, C. minutissimum, C. michailovskoense, andC. muscicola v.kashmiriense.— (6)Gomphosphaerioideae-species: Fifty eight populations (9 species) of the subfamilyGomphosphaerioideae (Microcystaceae, Chroococcales) from freshwater biotopes of Cuba were evaluated: Four planktic species with a probable cosmopolitan distribution were found (Coelosphaerium kuetzinginianum, C. minutissimum, Snowella lacustris andCoelomoron pusillus), and from the genusCoelomoron Buell two new species,C. microcystoides andC. vestitus were described. The tropical planktic speciesWoronichinia fremyi forms occasionally water blooms in larger reservoirs. Two tropical species from the genusGomphosphaeria Kütz, were recognized,G. multiplex (Nyg.) c. n. andG. semen-vitis sp. n.     

Studies on the Effect of Water-Soluble Polymers on Drug–Cyclodextrin Complex Solubility

The effect of complexation of irbesartan (IRB), a practically water-insoluble drug, with cyclodextrins in presence of different concentrations of water-soluble polymers (PEG 4000 and PVP K-90) on the dissolution rate of the drug has been investigated. Phase solubility studies were carried out to evaluate the solubilizing power of βCD in association with water-soluble polymers towards IRB and to determine the apparent stability constant (K _S) of the complexes. Improvement in K _S value for ternary complexes (IRB–βCD–polymers) clearly proved the benefit on the addition of water-soluble polymer to increase complexation efficiency. The dissolution rate of the drug from ternary systems containing PEG 4000 and PVP K-90 was higher as compared to the binary system. An optimum increase in the dissolution rate of the drug was observed at a polymer concentration of 5% w/w for PVP K-90 and 10% w/w for PEG 4000. DSC, FTIR, SEM, and XRD studies were carried out to characterize the complexes.     

Studies on the mechanism of estrogen biosynthesis in the rat ovary–I

Methods were evaluated for obtaining a reliable, active estrogen synthetase (aromatase) system from the rat ovary for mechanistic studies. Short terrn treatment with luteinizing hormone and follicle stimulating hormone in various combinations did not produce appreciable stimulation, whereas long term treatment (8–16 days) with pregnant mare's serum gonadotropin increased activity in homogenates up to nine fold per mg wet wt of tissue. A similar increase per mg protein was noted in the 105,000_g microsomal fraction where the bulk of the activity was found. Various conditions for preparing and incubating the aromatase were evaluated to obtain optimal enzyme activity. The potencies of six steroids as aromatase inhibitors were compared in the rat ovarian and human placental microsomal systems. In all cases except one the results were comparable.     

Studies on the oxidation of ω-hydroxyprostaglandins by an NAD-dependent dehydrogenase from mammalian liver cytosol

The oxidation of 12-hydroxylauric acid methyl ester (12-OH-L-Me) and of ω-hydroxy-prostaglandins (ω-OH-PGs) such as 20-OH-PGB₁ and 20-OH-PGE₁, was demonstrated with liver cytosol from rat, rabbit, and guinea pig in the presence of NAD; however, NADP did not support this oxidation. (ω-1)-Hydroxy-compounds (11-OH-laurate and 19-OH-PGB₁) and PGE₁, PGF_1α, and PGB₁, all lacking the terminal (ω)-hydroxyl, did not reduce NAD. However, at pH 10, PGE₁ slightly enhanced NAD reduction, suggesting that at this pH PGE₁, could be a substrate for 15-hydroxy-PG dehydrogenase (PGDH). The oxidation products from incubations of 12-OH-L-Me, 20-OH-PGB₁-Me, and 20-OH-PGE₁ with guinea pig liver cytosol were isolated and identified by gas chromatography/mass fragmentation spectrometry as being the corresponding dicarboxylic acids. In contrast to the liver cytosol, guinea pig kidney cytosol had only a minimal effect on NAD reduction by 12-OH-L-Me but nevertheless did support the stimulation of NAD reduction by PGE₁, and PGF_1α, but not by PGB₁, indicating the participation of kidney cytosolic PGDH in PGE₁ and PGF_1α oxidation and demonstrating that the oxidation of ω-OH to the carboxylic acid is not mediated by PGDH. Though the in vivo rate of oxidation of ω-OH-PGs has not been established, these results suggest that the urinary dicarboxylic-PG metabolites involve a multiple sequentialstep oxidation of PGs involving ω-hydroxylation by an NADPH-cytochrome P-450 system in the endoplasmic reticulum and the subsequent oxidation of the ω-OH by an NAD-dependent dehydrogenase in the cytosol.     

Studies on adenosine 3′,5′-monophosphate phosphodiesterase of human erythrocyte membranes

In this work we show the existence of cyclic AMP phosphodiesterase (EC 3.1.4.17) in human erythrocyte membranes and have clarified some properties of the enzyme. In human erythrocytes, about 23% of the total cyclic AMP phosphodiesterase activity is in a membrane-bound form. Although it could be solubilized with Triton X-100 in 5 mM Tris-HCl buffer (pH 8.0), it was not solubilized by a low or high concentration of salt. The enzyme seems to be localized in the cytoplasmic surface, since it is detected in sealed inside-out vesicles of human erythrocyte membranes, but not in intact human erythrocytes. The optimum pH was found to lie between 7.4 and 8.0, and Mg²⁺ was found to be necessary for its activity. Ca²⁺ and calmodulin could not stimulate the activity of this enzyme. Theophylline was a strong inhibitor, but cyclic GMP could not inhibit the enzymic hydrolysis of cyclic [³²P]AMP and this membrane-bound enzyme therefore seems to be specific to cyclic AMP.     

Studies on the Fluorescence of Saké

By spectrofluorophotometric investigation on various kinds of Saké it was found that they have at least two kinds of fluorescent colors, the one is blue, the other yellowish green. The former is always more dominant than the latter, but is unstable although the intensities of both color decrease remarkably by treatment of active charcoal. Ferulic acid and harman as the blue fluorescent components are isolated, the former from Saké in young, the latter from Saké kept for a long time under direct sun light.     

Studies of the effect of environmental factors on the rotifer predator–prey system in freshwater

The aim of this work is to evaluate the effect of environmental factors: temperature and photoperiod on the zooplankton predator–prey system. Rotifers, an important and cosmopolitan group of zooplankton in freshwater, were used in our study. We investigated the effect of temperature (20, 23, and 30°C) and of photoperiod (L:D = 12:0 and 0:12) on the predatory rotifer Asplanchna brightwelli consuming rotifer Brachionus calyciflorus as prey. Under A. brightwelli predation, populations of B. calyciflorus prey were consumed more slowly at 20 ± 1 and 30 ± 1°C as compared to 23 ± 1°C. Prey consumption by A. brightwelli increased from 0.63 ± 0.09 ind. predator⁻¹ at 20°C to a peak of 1.22 ± 0.12 ind. predator⁻¹ at 23°C, then decreased significantly to 0.93 ± 0.14 ind. predator⁻¹ at 30 ± 1°C. In addition, predation responded to temperature changing sensitively and rapidly. Statistical analysis showed that the prey consumption were significant different under altered temperature periods during 12 h. Photoperiod also significantly influenced the rate of A. brighwelli predation. B. calyciflorus suffered less predation in darkness than in light. The rate of prey consumption in light (1.06 ind. predator⁻¹) was twice the average of that in darkness (0.51 ind. predator⁻¹). Furthermore, predation rate varied under changing photoperiod but predators moved back into the light did not resume their original consumption rate. Our results demonstrate that whether the predation in rotifer successfully or not is strongly influenced by temperature and photoperiod.     

Studies on the thermostability of the α-amylase of Bacillus caldovelox

Summary Molecular mechanisms of thermoinactivation of the thermostable -amylase of Bacillus caldovelox were examined. Monomolecular conformational processes were found to be the major causes of thermoinactivation at both pH 4.5 and 8.0. The enzyme possessed considerable additional thermostability at pH 8.0, with half-lives of 0.75 and 7.0 min at 90° C and pH 4.5 and 8.0, respectively. The amino acid composition was examined with respect to the underlying thermostability exhibited by this enzyme. The inherent thermostability exhibited may be due to the high proline content (4.47 mol%), but more likely due to the high content of residues forming hydrophobic bonds (60.89 mol%) allied to a low content of residues responsible for ionic interactions (28.34 mol%). Offprint requests to: C. T. Kelly     

Theoretical studies on the reaction mechanism of PP1 and the effects of different oxidation states of the Mn–Mn center on the mechanism

Protein phosphatase 1 (PP1) is a dinuclear metalloenzyme that catalyzes the dephosphorylation of serine and threonine residues. In this work, the catalytic reaction mechanism of PP1 was theoretically investigated by hybrid density functional theory. Firstly, an initial model of the Mn(II)–Mn(II) active site of PP1 was constructed on the basis of the high-resolution crystal structure, and stationary points along the reaction pathway were optimized and analyzed. The calculations provide strong support for the mechanism of the dephosphorylation by PP1 and suggest that His125 plays the role of donating a proton to the leaving group. Furthermore, reaction models with the Mn–Mn centers at different oxidation states [Mn(III)–Mn(II) and Mn(III)–Mn(III) centers] were designed. Our calculations show that increasing the oxidation state of one or both Mn(II) can shorten the bond lengths between the metal ions and the ligands, and increase the energy barrier of the related reactions. We found it interesting that artificially adding a negatively charged hydroxy ligand into the Mn(III)–Mn(II) center can recover the shortened coordination bonds and lower the increased energy barrier. Our investigation suggests that the definite oxidation states of the metal centers should be significantly correlated to the negative charges of the ligands not only in phosphoprotein phosphatases, but also in purple acid phosphatases and Escherichia coli 5′-nucleotidase. This means that all the members of phosphoprotein phosphatases adopt homodivalent centers, and suggests the heterovalent active sites of purple acid phosphatases have evolved from homodivalent ones.     

Microscopical and Spectroscopic Studies on the Fluorescence of a Daunomycin–aluminum Complex

In this study, the spectroscopic features and microscopical applications of the fluorescent daunomycin-Al3+ complex have been analyzed. In the presence of Al3+, the absorption spectrum of daunomycin showed a deep bathochromic shift and new peaks at 529 and 566nm, whereas the fluorescence emission was considerably modified. The emission of daunomycin alone (peak at 560nm under optimal excitation at 470nm) decreased continuously from 0.5 to 24h after addition of Al3+ ions, and a new emission peak appeared at 580nm (optimal excitation at 530nm). Under the fluorescence microscope using green exciting light, nuclei from chicken blood smears and paraffin sections of rat embryos stained with daunomycin showed a weak emission, which greatly increased after treatment with Al3+ ions. The bright and stable fluorescence of chromatin DNA induced by daunomycin-Al3+ could be a valuable labelling method in fluorescence microscopy and DNA cytochemistry.     

Cell oxidation–reduction imbalance after modulated radiofrequency radiation

Abstract

Aim of this study was to evaluate an influence of modulated radiofrequency field (RF) of 1800?MHz, strength of 30?V/m on oxidation–reduction processes within the cell. The assigned RF field was generated within Gigahertz Transversal Electromagnetic Mode cell equipped by signal generator, modulator, and amplifier. Cell line V79, was irradiated for 10, 30, and 60?min, specific absorption rate was calculated to be 1.6?W/kg. Cell metabolic activity and viability was determined by MTT assay. In order to define total protein content, colorimetric method was used. Concentration of oxidised proteins was evaluated by enzyme-linked immunosorbent assay. Reactive oxygen species (ROS) marked with fluorescent probe 2′,7′-dichlorofluorescin diacetate were measured by means of plate reader device. In comparison with control cell samples, metabolic activity and total protein content in exposed cells did not differ significantly. Concentrations of carbonyl derivates, a product of protein oxidation, insignificantly but continuously increase with duration of exposure. In exposed samples, ROS level significantly (p?<?0.05) increased after 10?min of exposure. Decrease in ROS level was observed after 30-min treatment indicating antioxidant defence mechanism activation. In conclusion, under the given laboratory conditions, modulated RF radiation might cause impairment in cell oxidation–reduction equilibrium within the growing cells.     

Studies on the porphobilinogen deaminase–uroporphyrinogen cosynthetase system of cultured soya-bean cells

1. Porphobilinogenase was isolated and purified from soya-bean callus tissue; its components, porphobilinogen deaminase and uroporphyrinogen isomerase, were separated and purified. 2. The purified porphobilinogenase was resolved into two bands on starch-gel electrophoresis. The molecular weights of porphobilinogenase, deaminase and isomerase fractions were determined by the gel-filtration method. Porphobilinogenase activity was affected by the presence of air; uroporphyrinogens were only formed under anaerobic conditions, although substrate consumption was the same in the absence of oxygen as in its presence. 3. pH-dependence of both porphobilinogenase and deaminase was the same and a sharp optimum at pH 7.2 was obtained. Isomerase was heat-labile, but the presence of ammonium ions or porphobilinogen afforded some protection against inactivation. The action of several compounds added to the system was studied. Cysteine, thioglycollate, ammonium ions and hydroxylamine inhibited porphobilinogenase; certain concentrations of sodium and magnesium salts enhanced activity; some dicarboxylic acids and 2-methoxy-5-nitrotropone inhibited the deaminase. 4. delta-Aminolaevulate and ethionine in the culture media stimulated porphyrin synthesis and increased porphobilinogenase activity, whereas iron deficiency resulted in porphyrin accumulation. 5. The development of chlorophyll and porphobilinogenase on illumination of dark-grown callus was followed. 6. A hypothetical scheme is suggested for the enzymic synthesis of uroporphyrinogens from porphobilinogen.     

Effect of erythrocyte transfusion on the circulation of Guérin carcinoma-bearing rats

In Guérin carcinoma-bearing rats during tumour growth cardiac output and blood flow to several organs were increased whereas TPR, vascular resistance of some regions and haematocrit gradually decreased. In response to erythrocyte transfusion the anaemia of tumour-bearing rats was considerably improved or corrected, however, the parameters for systemic and regional circulation were only partially restored. We presume that the circulatory "hyperkinesis" of tumour-bearing rats is partly due to the anaemia but also an unknown factor must be considered. The blood perfusion of the Guérin carcinoma was not affected by the erythrocyte transfusion thus it is feasible that the tumour vasculature is maximally dilated even with a normal haematocrit of the host.     

Studies of the MHC-linked “CT” alloantigenic systems in rats

Cytotoxic T lymphocyte (CTL) populations from Lewis rats were generated by combined immunization in vivo and restimulation in vitro with lymphoid cells from Fischer 344 donors. This strain combination is phenotypically identical at the MHC (Ag-B) by serologic, MLC, and GVH criteria, and thus, it is to be expected that CTL raised in this combination would show MHC restricted cytolysis against the existing minor alloantigenic differences. Previous studies demonstrated instead that CTL are effective against target cells from a variety of strains, regardless ofMHC haplotype, and that these cytotoxic T cell defined target antigens (CT antigens) are controlled by genes which map with the MHC. The present studies show that the CT antigens defined by LF CTL are unlike gene products expected for eitherSD-like orLD-like region genes, and it is therefore concluded that they mark a novel subregion of the rat MHC or are specified by a locus (loci) linked to, but outside MHC.Supported by U.S.P.H. Grants CA-15822, CA-09140, and AI-10961     

Studies on energy-linked reactions. Energy-linked reduction of oxidized nicotinamide–adenine dinucleotide by succinate in Escherichia coli

1. A method for the preparation of small particles from Escherichia coli is described. 2. These particles catalyse the ATP-driven reduction of NAD⁺ by succinate. 3. ATP utilized/NADH produced ratios below 2.0 were measured and a stoicheiometry of 1 molecule of ATP utilized per molecule of NADH produced is proposed. 4. The reaction is not inhibited by 2,4-dinitrophenol or by oligomycin but is inhibited by other uncouplers such as pentabromophenol and dicoumarol. 5. The specificity of the energy source, the specificity of the electron acceptor, the effects of pH, Mg²⁺, P_i and temperature have been studied.     

Lipogenesis in the brain of suckling rats. Studies on the mechanism of mitochondrial–cytosolic carbon transfer

1. Studies on the incorporation of [3-¹⁴C]pyruvate and d-3-hydroxy[3-¹⁴C]butyrate into the brain lipid fraction by brain homogenates of the suckling (7-day-old) rat have been carried out. 2. Whereas approximately twice as much CO₂ was evolved from pyruvate compared with 3-hydroxybutyrate metabolism, similar amounts of the radioactivity of these two precursors were incorporated into the lipid fraction. Furthermore, in both cases the incorporation into lipid was almost tripled when glucose (10mm) or NADPH (2.5mm) was added to the incubation media. 3. If 5mm-(—)-hydroxycitrate, an ATP–citrate lyase inhibitor, was added to the incubation the incorporation of carbon from pyruvate was inhibited to 39% of the control and from 3-hydroxybutyrate to 73% of the control, whereas CO₂ production from both precursors was not affected. 4. The incorporation from pyruvate or 3-hydroxybutyrate into lipids was not affected by the presence of 10mm-glutamate in the medium (to encourage N-acetylaspartate production). However, incorporation from pyruvate was inhibited by 21% in the presence of 5mm-amino-oxyacetate (a transaminase inhibitor) and by 83% in the presence of both hydroxycitrate (5mm) and amino-oxyacetate. 5. Incorporation from 3-hydroxybutyrate into brain lipids was inhibited by 20% by amino-oxyacetate alone, but by 55% in the presence of both hydroxycitrate and amino-oxyacetate. 6. It is concluded that the mechanism of carbon transfer from pyruvate into lipids across the mitochondrial membrane in the suckling rat brain is mainly via citrate and N-acetylaspartate. 3-Hydroxybutyrate, in addition to using these routes, may also be incorporated via acetoacetate formation and transport to the cytosol.