Bacteriochlorophyll c monomers,dimers, and higher aggregates in dichloromethane,chloroform, and carbon tetrachloride

Bacteriochlorophyll c in vivo is a mixture of at least 5 homologs, all of which form aggregates in CH₂Cl₂, CHCl₃ and CCl₄. Three homologs exist mainly in the 2-R-(1-hydroxyethyl) configuration, whereas the other two homologs, 4-isobutyl-5-ethyl and 4-isobutyl-5-methyl farnesyl bacteriochlorophyll c, exist mainly in the 2-S-(1-hydroxyethyl) configuration (Smith KM, Craig GW, Kehres LA and Pfennig N (1983) J. Chromatograph. 281: 209–223). In CCl₄ the S-homologs form an aggregate of 2–3 molecules whose absorption (747 nm maximum) and circular dichroism spectra resemble those of the chlorosome. In CH₂Cl₂, CHCl₃ and CCl₄ the 4-n-propyl homolog (R-configuration) forms dimers absorbing at ca. 680 nm and higher aggregates absorbing at 705–710 nm. In CCl₄ the dimerization constant is approx. 10 µM^–1 (1000 times that for chlorophyll a). The difference between the types of aggregates formed by the 4-n-propyl and 4-isobutyl homologs is attributed to the difference between the R- and S-configurations of the 2-(1-hydroxyethyl) groups in each chlorophyll.Abbreviations BChl bacteriochlorophyll - CD circular dichroism - Chl chlorophyll - DNS data not shown - EEF 4-ethyl-5-ethyl farnesyl - iBM/EF 4-isobutyl-5-methyl/ethyl farnesyl - MEF 4-methyl-5-ethyl farnesyl - PEP 4-n-propyl-5-ethyl farnesyl     

Protective effect of bixin on carbon tetrachloride-induced hepatotoxicity in rats

Background

The liver is an important organ for its ability to transform xenobiotics, making the liver tissue a prime target for toxic substances. The carotenoid bixin present in annatto is an antioxidant that can protect cells and tissues against the deleterious effects of free radicals. In this study, we evaluated the protective effect of bixin on liver damage induced by carbon tetrachloride (CCl₄) in rats.

Results

The animals were divided into four groups with six rats in each group. CCl₄ (0.125 mL kg^-1 body wt.) was injected intraperitoneally, and bixin (5.0 mg kg^-1 body wt.) was given by gavage 7 days before the CCl₄ injection. Bixin prevented the liver damage caused by CCl₄, as noted by the significant decrease in serum aminotransferases release. Bixin protected the liver against the oxidizing effects of CCl₄ by preventing a decrease in glutathione reductase activity and the levels of reduced glutathione and NADPH. The peroxidation of membrane lipids and histopathological damage of the liver was significantly prevented by bixin treatment.

Conclusion

Therefore, we can conclude that the protective effect of bixin against hepatotoxicity induced by CCl₄ is related to the antioxidant activity of the compound.     

Confirmation of assignment of the trichloromethyl radical spin adduct detected by spin trapping during 13C-carbon tetrachloride metabolism in vitro and in vivo

Rat liver microsomal incubation systems containing the free radical spin trap, phenyl-t-butyl nitrone, as well as an NADPH generating system and [¹³C]CCl₄ (90 atom % ¹³C) produce electron spin resonance spectra consistent with that expected for a trichloromethyl-phenyl-t-butyl nitrone adduct. This same spectrum is observed in a lipid extract of the liver from a rat orally administered [¹³C]CCl₄ as well as in a solution of phenyl-t-butyl nitrone and [¹³C]CCl₄ irradiated with ultraviolet light.     

Infra-red spectroscopic study for decalcification in the dentine

Summary A study was made of spectra of decalcified human dentine being treated with five decalcifying agents to investigate the differences of infrared spectra of decalcified dentine.Solutions of 20% 4-Na-EDTA, 5% HNO₃, 10% sulfosalicylic acid, 10% CCl₃COOH and 10% lithium lactate were used as decalcifying agents. Infrared absorption spectra between 1800 and 650 cm^–1 were obtained for the investigation of the absorption strength at 1033,1095; 1412–1414 to 1450–1455; 875–870; 1649 and 1550 cm^–1 before and after decalcification.The specimens following decalcification with EDTA and HNO₃ showed almost no influences to the absorption strength at 1649 and 1550 cm^–1 of amides. Such absorption spectra were relatively slight in CCl₃COOH and sulfosalicylic acid. The absorption strength at 1033 and 1095 cm^–1 bands of PO ₄ ^––– may indicate a standard of decalcification. Quick and complete decalcification of dentine was performed by HNO₃, CCl₃COOH and sulfosalicylic acid but slower and more incomplete decalcification resulted with EDTA and lithium lactate.     

Respiratory Transition during Seed Germination

Experiments with germinating seeds of Wayne soybean (Glycine max Merr.) show that between the 4th and the 8th hour of germination, respiration experiences a transition from predominantly “alternate” respiration, which is sensitive to salicylhydroxamic acid, to a cyanide-sensitive respiration. The dependence of early germination stages on alternate respiration is reflected in several types of seed functions, including subsequent root growth rate, chlorophyll synthesis, and germination itself. The early period of germination is shown to require a normal O₂ tension, which is no longer a requirement at later stages. The changing sensitivity to cyanide and to salicylhydroxamic acid is found to be common to seven different types of germinating seeds. It is proposed that the alternate pathway of respiration provides something essential for the completion of the earliest stages of seed germination.     

Cold tolerance in CCl4-treated rats and its modification by administration of garlic oil and glucose

Male Wistar rats weighing 150–200 g maintained under standard laboratory conditions and given Hindustan Lever Pellets and waterad libitum were exposed to –20°C for determination of the rate of fall of rectal temperature and survival time. The rate of fall of body temperature was significantly increased and the survival time was reduced, when animals were given an intraperitoneal injection of 1 ml/kg BW of CCl₄ 24 h but not 2 h earlier. Pre-treatment of the animals with 0.006 ml of garlic oil in a 2% solution of arachis oil for 3 days gave a significant protection to the animals against the CCl₄-induced fall in cold tolerance. Administration of glucose orally 300 mg in 2 ml of saline eliminated the CCl₄-induced fall in cold tolerance. The animals displayed a hypoglycemia 24 h, but not 2 h after injection of CCl₄. CCl₄-induced hypoglycemia was reduced by pre-treatment with garlic oil. The results indicate that the CCl₄-induced reduction in cold tolerance is secondary to hypoglycemia and not due to the direct effect of CCl₄ on the thermoregulatory mechanism in the CNS. The critical level of blood glucose below which the cold tolerance is reduced was found to be 76 mg/100 ml of blood.     

Low temperature spectral properties of subchloroplast fractions purified from spinach

Spinach (Spinacia oleracea L.) chloroplasts solubilized by digitonin were separated into five fractions by sucrose density gradient centrifugation. Three of the fractions, F_I, F_II, and F_III, corresponding to photosystem I, photosystem II, and the chlorophyll a/b complex, were purified further by two steps of diethylaminoethyl-cellulose chromatography followed by electrofocusing on an Ampholine column. The polypeptide patterns of the fractions were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the spectral properties of the fractions at −196 C determined by absorption spectra, fourth derivative curves of the absorption spectra, fluorescence emission spectra, and fluorescence excitation spectra. The activity of purified F_II (photosystem II) was also assayed by the photoreduction of dichlorophenol-indophenol at room temperature using 1,5-diphenylcarbohydrazine as the electron donor and by the photoreduction of C-550 at −196 C. The different fractions showed unique polypeptide patterns and unique sets of low temperature-absorbing forms of chlorophyll. The fluorescence emission spectra of F_I, F_II, and F_III at −196 C were also unique with maxima at 734, 685 and 681 nm, respectively. F_I showed negligible emission at wavelengths shorter than 700 nm and the long wavelength tails of F_II and F_III in the 730 nm region were relatively small (approximately 10% of emission of their wavelength maxima). Addition of 0.1% Triton to F_I and F_II caused the longer wavelength absorbing forms of chlorophyll to shift to 670 nm and the fluorescence emission maxima (of both fractions) to shift to 679 nm at −196 C with an increase in the yield of fluorescence especially in the case of F_I.     

Histochemical Compartmentation of Photosynthesis in the Crassulacean Acid Metabolism Plant Crassula falcata

The succulent leaf of the obligate Crassulacean acid metabolism plant Crassula falcata comprises two distinct types of parenchyma. The peripheral tissue is dark green, whereas the central tissue is relatively colorless. We have investigated whether the conventional interpretation of Crassulacean acid metabolism—simply, temporal separation of light and dark CO₂ fixation within individual cells—is sufficient. Ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) and chlorophyll, indicating the photosynthetic-carbon-reduction pathway, were concentrated in peripheral tissue. Specific activities of P-enolpyruvate carboxylase (4.1.1.31) and of NAD⁺-malic enzyme (1.1.1.39), indicating capacity for dark CO₂ fixation and release, respectively, were high in both types of parenchyma. Measured directly as malic acid decline at the beginning of the photoperiod, CO₂ “storage” occurred in both tissues. These data indicate that there is a spatial component to Crassulacean acid metabolism in C. falcata.     

Inhibitory Effect of Gallic Acid on CCl4-Mediated Liver Fibrosis in Mice

The aim of this study was to investigate the effect of gallic acid (GA) on liver fibrosis induced by carbon tetrachloride (CCl₄). Male BALB/c mice were randomly divided into four groups: normal control group (group A), CCl₄-induced liver injury control group (group B), and CCl₄ induction with GA of low dose (5 mg/kg) and high dose (15 mg/kg) treatment group (group C and group D). GA was intra-gastric given for mice once a day after 2 weeks of CCl₄ induction. Animals were killed at the eighth week. Degrees of fibrosis and collagen percentage were measured. Hyaluronic acid (HA), type IV collagen (cIV), malondialdehyde (MDA), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and gamma-glutamyl transferase (γ-GT) were determined. Expression of matrix metalloproteinases-2 (MMP-2) and tissue inhibitor of matrix metalloproteinases-1 (TIMP-1) mRNA levels were examined by RT-PCR. Western blotting was carried out to evaluate the changes of MMP-2 protein. HE and VG stainings showed GA in a dose-dependent manner improved significantly the fibrosis condition in CCl₄-injured mice (P < 0.05 or P < 0.01). Also, the concentrations of HA, cIV, and MDA, as well as the serum levels of ALT, AST, and γ-GT were markedly reduced by GA (P < 0.05 or P < 0.01), and decreases in MMP-2, TIMP-1 mRNA, and MMP-2 protein were observed as well (P < 0.05 or P < 0.01). GA could exert protective effect on liver injury and reduce liver fibrosis induced by CCl₄ in mice, which might be through the inhibition of hepatic stellate cell activity.     

Photoinactivation of Catalase Occurs under Both High- and Low-Temperature Stress Conditions and Accompanies Photoinhibition of Photosystem II

Severe photoinactivation of catalase (EC 1.11.1.6) and a decline of variable fluorescence (F_v), indicating photoinhibition of photosynthesis, were observed as rapid and specific symptoms in leaves exposed to a high heat-shock temperature of 40°C as well as in leaves exposed to low chilling temperatures in white light of only moderately high photosynthetic photon flux density of 520 μE m⁻² s⁻¹. Other parameters, such as peroxidase (EC 1.11.1.7), glycolate oxidase (EC 1.1.3.1), glutathione reductase (EC 1.6.4.2), or the chlorophyll content, were hardly affected under these conditions. At a compatible temperature of 22°C, the applied light intensity did not induce severe photoinactivations. In darkness, exposures to high or low temperatures did not affect catalase levels. Also, decline of F_v in light was not related to temperature sensitivity in darkness. The effective low-temperature ranges inducing photoinactivation of catalase differed significantly for chilling-tolerant and chilling-sensitive plants. In leaves of rye (Secale cereale L.) and pea (Pisum sativum L.), photoinactivation occurred only below 15°C, whereas inactivation occurred at 15°C in cucumber (Cucumis sativus L.) and maize (Zea mays L.). The behavior of F_v was similar, but the difference between chilling-sensitive and chilling-tolerant plants was less striking. Whereas the catalase polypeptide, although photoinactivated, was not cleaved at 0 to 4°C, the D1 protein of photosystem II was greatly degraded during the low-temperature treatment of rye leaves in light. Rye leaves did not exhibit symptoms of any major general photodamage, even when they were totally depleted of catalase after photoinactivation at 0 to 4°C, and catalase recovered rapidly at normal temperature. In cucumber leaves, the decline of catalase after exposures to bright light at 0 to 4°C was accompanied by bleaching of chlorophyll, and the recovery observed at 25°C was slow and required several days. Similar to the D1 protein of photosystem II, catalase differs greatly from other proteins by its inactivation and high turnover in light. Inasmuch as catalase and D1 protein levels depend on continuous repair synthesis, preferential and rapid declines are generally to be expected in light whenever translation is suppressed by stress actions, such as heat or chilling, and recovery will reflect the repair capacity of the plants.     

Partial analysis of the bands seen in circular dichroism spectra of green and blue-green algal cells and thylakoids

A comparison was made of the circular dichroism (C.D.) spectra of Chlorella, Euglena, and Anacystis cells and thylakoids. Analyses of the spectra reveal that these C.D. bands are similar to those observed previously in whole spinach choloroplasts and subchloroplast particles. C.D. spectra of Euglena chloroplasts show bands at longer wavelengths than previously reported. From comparisons of circular dichroism spectra and fine structure, it was concluded that: (a) bands seen in circular dichroism spectra were not the result of light scattering from thylakoid membranes; and (b) bands seen in the C.D. spectra of nonmembranous systems (previously reported) could account for circular dichroism of algae. We also concluded that comparisons would have to be made with model systems in order to correct for effects of absorption flattening, concentration obscuring, and differential light scattering of membranous systems.     

The action of lipases on chloroplast membranes. III. The effect of lipid hydrolysis on chlorophyll-protein complexes in thylakoid membranes

Bean thylakoid membranes treated with various lipolytic enzymes (bean galactolipase, phospholipases A₂, C, D) showed marked changes in their acyl lipid composition. As a consequence of acyl lipids hydrolysis, destruction of some chlorophyll a-protein complexes (CP1a, CP1, CPa) or monomerization of the oligomeric of light harvesting chlorophyll a/b protein complex (LHCP) was observed. It is concluded that galactolipids and phosphatidylcholine are responsible for the stability of CP1a, CP1 and CPa, respectively. Phosphatidylglycerol and to some extent monogalactosyldiacylglycerol are essential for the stabilization of oligomeric structures of light harvesting chlorophyll a/b protein complex.Abbreviations chl chlorophyll - CP1a, CP1 chl a-protein complexes, of PSI - CPa chl a-protein complex of PSII - DGDG diagalactosyldiacylglycerol - FC free chl - GL galactolipase - LHCP^1–3 light harvesting chl a/b protein complex - MGDG monogalactosyldiacylglycerol - PAGE polyacrylamide gel electrophoresis - PC phosphatidylcholine - PG phosphatidylglycerol - PLA₂ phospholipase A₂ - PL phospholipase C - PLD phospholipase D - PSI photosystem I - PSII photosystem II - SDS sodium dodecyl sulphate - SQDG sulfoquinovosyl-diacylglycerol - TCA trichloroacetic acid - Tricine N-tris-(hydroxymethyl)-methylglycine - Tris Tris-(hydroxymethyl)-aminomethan     

Monomers,dimers, and tetramers of 4-n-propyl-5-ethyl farnesyl bacteriochlorophyll c in dichloromethane and carbon tetrachloride

Absorption (ABS) and circular dichroism (CD) spectra were recorded for 6 concentrations (2.0–290 M) of bacteriochlorophyll (BChl) c in each solvent. Monomer spectra were obtained by adding methanol (1:200) to each sample. The monomer showed an ABS peak and a CD trough at 664 nm in CH₂Cl₂ (ABS peak at 665 nm in CCl₄). Dimer-plus-monomer spectra were obtained by subtracting high concentration (e.g., 290 M) spectra appropriately scaled from lower concentration (e.g., 26 M) spectra. Pure dimer spectra were then obtained by subtracting monomer spectra appropriately scaled from dimer-plus-monomer spectra. The dimer showed an ABS peak at 679 nm in both CH₂Cl₂ and CCl₄ and a CD trough at ca. 670 nm in CH₂Cl₂. The optical properties of the dimer do not agree with the model for bacteriochlorophyllide d [Smith KM, Bobe FW, Goff DA and Abraham RJ (1986) J Am Chem Soc 108: 1111–1120]. Higher aggregate spectra were obtained by subtracting appropriately scaled monomer and dimer spectra from high concentration (e.g., 290 M) spectra. The aggregate showed ABS shoulders at ca. 636 and 678 nm with a peak at 702 nm in CH₂Cl₂ and at 708 nm in CCl₄; the CD spectrum in either solvent showed peaks at 638 and 679 nm with troughs at 658 and ca. 710 nm. These spectra are consistent with an excitonic interaction between 4 chromophores in the aggregate. Each of the 12 original ABS spectra was deconvoluted in terms of the appropriate monomer, dimer and aggregate spectra, and the concentrations of each component were determined. Plots of log aggregate concentration vs. log dimer concentration lay on or near a line of slope 1.9 for CH₂Cl₂ and on or near a line of slope 2.1 for CCl₄. The aggregate was thus shown to be a tetramer. The theoretical relationship between dimers and monomers (slope 2.0) was not observed in all cases.Abbreviations ABS absorbance - BChl bacteriochlorophyll - CD circular dichroism - Chl chlorophyll - DNS data not shown - PEF 4-n-propyl-5-ethyl farnesyl     

Isolation of Mesophyll Cells from Sedum telephium Leaves

A technique is described for mechanically isolating metabolically active individual spongy mesophyll cells from the Crassulacean acid metabolism plant, Sedum telephium. Mature leaves are selected at about 2 PM when acidity is low, and three different media are used to reduce the problem of leaf acidity and to maintain isotonic conditions. The upper and lower epidermis is peeled from chilled leaves and the leaves are suspended in a buffered “soaking medium,” then gently ground with a mortar and pestle. Cells and debris are separated using a “washing medium,” with cells being filtered through a 136 micron net and collected on an 80 micron net. Cells then are suspended in a “cell suspension medium” and concentrated by centrifugation. Approximately 2 hours are required for the isolation procedure, and activity in CO₂ fixation is constant for up to 4 hours after isolation. Microscopic examination shows about 65% of the isolated cells appear intact and unplasmolyzed and are similar to leaf msophyll cells. The yield of cells on a leaf chlorophyll basis is about 1%. A light micrograph of the isolated cells is given.     

Topological Analysis of DcuA, an Anaerobic C4-Dicarboxylate Transporter of Escherichia coli

Escherichia coli possesses three independent anaerobic C₄-dicarboxylate transport systems encoded by the dcuA, dcuB, and dcuC genes. The dcuA and dcuB genes encode related integral inner-membrane proteins, DcuA and DcuB (433 and 446 amino acid residues), which have 36% amino acid sequence identity. A previous amino acid sequence-based analysis predicted that DcuA and DcuB contain either 12 or 14 transmembrane helices, with the N and C termini located in the cytoplasm or periplasm (S. Six, S. C. Andrews, G. Unden, and J. R. Guest, J. Bacteriol. 176:6470–6478, 1994). These predictions were tested by constructing and analyzing 66 DcuA-BlaM fusions in which C terminally truncated forms of DcuA are fused to a β-lactamase protein lacking the N-terminal signal peptide. The resulting topological model differs from those previously predicted. It has just 10 transmembrane helices and a central, 80-residue cytoplasmic loop between helices 5 and 6. The N and C termini are located in the periplasm and the predicted orientation is consistent with the “positive-inside rule.” Two highly hydrophobic segments are not membrane spanning: one is in the cytoplasmic loop; the other is in the C-terminal periplasmic region. The topological model obtained for DcuA can be applied to DcuA homologues in other bacteria as well as to DcuB. Overproduction of DcuA to 15% of inner-membrane protein was obtained with the lacUV5-promoter-based plasmid, pYZ4.     

Hepatoprotective effect of bis(4‐methylbenzoyl) diselenide against CCl4‐induced oxidative damage in mice

From a pharmacological point of view, organoseleniums are compounds with important and interesting antioxidant and biological activities. The aim of this study was to evaluate the hepatoprotective effect of bis(4‐methylbenzoyl) diselenide (BMD) against carbon tetrachloride (CCl₄)–induced oxidative damage in mice. The animals received BMD (25 mg/kg p.o., for 3 days), and after 1 day, CCl₄ (1 mg/kg body weight) was administered by intraperitoneal route. One day after the CCl₄ exposure, the animals were euthanized for biochemical and histological analysis. Treatment with BMD (25 mg/kg p.o.) protected against aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, gamma‐glutamyl transferase and lactate dehydrogenase activity increases induced by CCl₄ plasma exposure. Treatment with BMD (25 mg/kg) protected against increases in thiobarbituric reactive species and decreasing non‐protein thiols and ascorbic acid levels in liver of mice. Catalase and superoxide dismutase activity inhibition in the liver caused by CCl₄ were protected by treatment with BMD (25 mg/kg). Glutathione S‐transferase activity was inhibited by CCl₄ and remained unaltered even after treatment with BMD. Sections of liver from CCl₄‐exposed mice presented an intense infiltration of inflammatory cells and loss of the cellular architecture. BMD (25 mg/kg) attenuated CCl₄‐induced hepatic histological alterations. The results demonstrated the hepatoprotective effects of BMD in the mouse liver, possibly by modulating the antioxidant status. Copyright © 2012 John Wiley & Sons, Ltd.     

Thermal Dependence of the Apparent K(m) of Glutathione Reductases from Three Plant Species

The thermal dependencies of the apparent K_m of the glutathione reductases from spinach (Spinacia oleracea L.) corn (Zea mays L.), and cucumber (Cucumis sativus L.) were determined. The apparent K_m of the enzymes were found to vary up to 9-fold between 12.5 and 45°C. Values of the apparent K_m in excess of 200% of the observed minimum are suggested to be detrimental to the normal function of the enzyme. We propose the term “thermal kinetic window” to describe to the range of temperatures over which the apparent K_m of the glutathione reductase is within 200% of its minimum and suggest that it may be a useful indicator of the limits of thermal stress for a given species. The thermal kinetic windows determined in this study are: <16°C for spinach, 23 to 32°C for corn, and 35 to 41°C for cucumber.     

The interaction of CCl<Subscript>4</Subscript> with Ng (Ng = He,Ne, Ar), O<Subscript>2</Subscript>, D<Subscript>2</Subscript>O and ND<Subscript>3</Subscript>: rovibrational energies,spectroscopic constants and theoretical calculations

This investigation generated rovibrational energies and spectroscopic constants for systems of CCl₄ with Ng (Ng?=?He, Ne, Ar), O₂, D₂O and ND₃ from scattering experimental data, and the results presented are of interest for microwave spectroscopy studies of small halogenated molecules. The rovibrational spectra were obtained through two different approaches (Dunham and DVR) within the improved Lennard Jones (ILJ) model. Spectra were also generated within ordinary Lennard Jones and deviations suggest that the ILJ model should be preferred due to interactions beyond dispersion forces presented in these systems. Data from the literature and additional high level quantum mechanical calculations presented in this work show that these systems should not be considered as van der Waals complexes due to halogen bonding (HB) interactions, and this is especially true for the CCl₄–D₂O and CCl₄–ND₃ complexes. The charge displacement from the latter systems are one order of magnitude higher than the values from literature for CCl₄ and He, Ne, Ar and O₂ systems, and show significant deviations between DFT and Hartree-Fock values not previously reported in the literature.     

Appearance of an Alternate Pathway Cyanide-resistant during Germination of Seeds of Cicer arietinum

The combined action of the inhibitors antimycin A and cyanide with benzohydroxamic acid indicates the presence of a cyanide-resistant pathway of respiration in chick pea (Cicer arietinum L.) seeds. The appearance of this pathway takes place during germination. During the first 12 hours of germination, the respiration is predominantly cyanide-sensitive, showing after this time a shift to an “alternate” respiration which is sensitive to benzohydroxamic acid, reaching the maximal cyanide resistance between 72 and 96 hours of germination. The appearance of the alternate pathway is initiated by high O₂ concentrations and depends on cytoplasmic protein synthesis, since its appearance is inhibited by cycloheximide but not by chloramphenicol. Actinomycin D has no effect on the appearance of the alternate pathway. Our results indicate, in agreement with other authors, that the branching point is located between the flavoproteins and cytochromes b, probably at the level of ubiquinone, but the possibility of more than one branching point of the electron flow is also considered.