Spectrophotometric characteristics and assay of pure pyronin Y

Summary The spectrophotometric characteristics of analytically pure pyronin Y have been investigated. Addition of metal ions (Fe³⁺, Zn²⁺, and Mg²⁺) and of dextrin were shown not to influence the absorption characteristics. The composition of the solvent strongly influenced the value of the extinction coefficient. Aqueous ethanolic solutions with a content of about 50% ethanol gave higher -values than those found for more concentrated ethanol solutions. The difference can be explained by the existence of a solvent-solute complex in the less concentrated ethanol solutions. A new spectrophotometric assay is proposed using the -value 11.7×10⁴ lmol^–1 cm^–1 found in aqueous ethanol (52%) as standard.     

Partial purification and characterization of the organic solvent-tolerant lipase produced by Pseudomonas fluorescens RB02-3 isolated from milk

Eighty-five putative Pseudomonas isolates were obtained from various raw milk and pasteurized milk samples using Pseudomonas CFC agar. Among them, 36 isolates were identified as Pseudomonas fluorescens, and one isolate was identified as Pseudomonas putida. Lipase activity of the strains was quantitatively measured by the spectrophotometric method using p-nitrophenyl palmitate (p-NPP) as substrate. Detected lipase activity of the strains was between 10.03 U/mL and 22.16 U/mL. Pseudomonas fluorescens RB02-3 possessed the highest lipase activity. The extracellular lipase of P. fluorescens RB02-3 strain was homogeneously purified using a combination of ammonium sulfate precipitation, dialysis, and gel filtration column chromatography. This purification procedure resulted in 2.97-fold purification with 20.3% recovery. The enzyme was characterized, and exhibited maximum activity at pH 7.0 and 50 °C; after it was incubated for 1 h it was activated in the presence of hexane, ethyl acetate, isopropanol, and ethanol and remained stable after the incubation was extended for 2 hr. The lipase was slightly inhibited in the presence of Zn2+, Co2+, Cu2+, Ni2+ salts, and ethylenediamine tetraacetic acid (EDTA), whereas Cd2+, sodium dodecyl sulfate (SDS), and Tween-80 had no effect on its activity.     

Hyperosmotic oxidation of glucose and decarboxylation of histidine in the gastric mucosa.

Paired slices of rat gastric mucosa were incubated with labeled glucose or histidine in isosomotic solution of 3-fold hyperosmotic solutions concentrated in NaCl, KCl, or ethanol. The rate of (1-14C)glucose oxidation to 14CO2 in isosmotic solution was reduced by 74% in hyperosmotic NaCl and by 28% in hyperosmotic KCl. The rate of (6-14C)glucose oxidation to 14CO2 in isosmotic solution was reduced by 64% in hyperosmotic NaCl and by 53% in hyperosmotic KCl. Reductions of glucose oxidation in hyperosmotic ethanol were not significant. The ratio of 14CO2 formed from (1-14C)glucose to that formed from (6-14C)glucose was not significantly changes by hyperosmotic NaCl or ethanol, but was significantly raised by hyperosmotic KCl. The rate of (carboxyl-14C)histidine decarboxylation in isosomotic solution was reduced significantly by 48% in hyperosmotic NaCl, by 30% in hyperosmotic KCl, and by 27% in hyperosmotic ethanol. We conclude that hyperosmotic solutions reduce glucose oxidation and histidine decarboxylation by rat gastric mucosa in the order of potency: NaCl greater than Kcl greater than or equal to ethanol. Thus hyperosmotic solutions inhibit the source of metabolic energy for stimulated acid secretion, the citric acid cycle, and the formation of the secretagogue histamine.     

Transitions of poly(dI-dC), poly(dI-methyl5dC) and poly(dI-bromo5dC) among and within the B-, Z-, A- and X-DNA families of conformations.

It is shown, using circular dichroism spectroscopy, that poly(dI-dC) is capable to isomerize into both Z-DNA and A-DNA in concentrated NaCl + NiCl2 and trifluoroethanol solutions, respectively. This polynucleotide also undergoes a cooperative, two-state transition in ethanol into a structure which most probably is a canonical B-DNA. This implies that the conformation of poly(dI-dC) is unusual in low-salt aqueous solution. The canonical B-DNA is also adopted by poly(dI-methyl5dC) in trifluoroethanol while this polynucleotide adopts Z-DNA not only in NaCl + NiCl2 but also in the presence of MgCl2. Poly(dI-methyl5dC) partially adopts X-DNA in concentrated CsF and mainly ethanolic solutions. Poly(dI-bromo5dC) isomerizes into Z-DNA not only in concentrated NaCl even in the absence of NiCl2 but also in concentrated MgCl2. This polynucleotide transforms between two distinct variants of Z-DNA in ethanol or trifluoroethanol solutions.     

Oxygen equilibrium of emulsified solutions of normal and sickle hemoglobin.

Emulsions containing microdroplets of concentrated solutions of normal or sickle hemoglobin dispersed in a continous oil phase have been prepared, and the aggregation of sickle hemoglobin within microdroplets in a deoxygenated emulsion demonstrated. The equilibrium oxygen saturation of hemoglobin in the emulsions has been measured as a function of the partial pressure of oxygen by a novel spectrophotometric technique which corrects for the scattering of light in the emulsion. The half-saturation oxygen pressure and cooperativity of oxygen binding are substantially greater in concentrated (27 g/dl) solutions of sickle hemoglobin than in solutions of non-aggregating hemoglobin. The shape of the oxygen equilibrium curve of concentrated sickle hemoglobin is qualitatively discussed in terms of a previously proposed model (1).     

Isomeric equilibria in complexes of adenosine 5'-triphosphate with divalent metal ions. Solution structures of M(ATP)2- complexes

Solution structures of M(ATP)2- complexes are reviewed. First the self-stacking properties of ATP4- and M(ATP)2- are shortly described. It is emphasized that for an evaluation of solution structures of M(ATP)2- complexes only results from diluted solutions (below 1 mM) should be used. Next, a comprehensive set of stability data obtained under such conditions from potentiometric pH titrations is summarized for the complexes of Mg2+, Ca2+, Mn2+, Co2+, Ni2+, Cu2+, Zn2+ and Cd2+ with ATP, and for comparison also with pyrimidine nucleoside 5'-triphosphates (YTPs), i.e. CTP, UTP and TTP. The stabilities for the M(ATP)2- complexes are mostly larger than those for the corresponding M(YTP)2- species; this increased stability results from the metal ion back-binding to the base residue in M(ATP)2-, i.e. macrochelates are formed. Detailed analysis of the stability data allows calculation of the percentage of the closed form for the several M(ATP)2- complexes: back-binding is most pronounced in Cu(ATP)2- (67 +/- 2%), remarkable in Zn(ATP)2- (28 +/- 7%), and not observable for Ca(ATP)2- (2 +/- 6%). Comparison of these results with those from 1H-NMR and ultraviolet spectrophotometric studies allows the conclusion that two types of base back-bound macrochelates are formed: one with a direct, i.e. innersphere, M2+/N-7 coordination, and one with a water molecule between the metal ion and N-7, i.e. an outersphere interaction occurs [e.g. to about 10% in Mg(ATP)2-] through hydrogen bonding of a coordinated water to N-7. The formation degree of both forms of these closed isomers is quantified. The biological implications of these results are indicated and the versatility of ATP as a ligand is discussed by summarizing pertinent examples.     

Determination of the physicochemical constants and spectrophotometric characteristics of the metallochromic Zincon and its potential use in biological systems

In this work we report a detailed characterization of the metallochromic Zincon. Zincon forms complexes with Zn2+ and Cu2+, producing change in colour; the complexes with Fe2+, Mn2+ and Ca2+ cause the bleaching of the Zincon solutions. Mg2+ does not interact with Zincon nor does it change its spectral characteristics. The presence of Ca2+ and Mg2+ does not interfere with the spectral characteristics of the Zn-Zincon complex. The Kd, Ks and delta epsilon values for the complexes were determined. The delta epsilon values were very high, making this spectrophotometric method very sensitive. The complex Zn-Zincon is fully reversible; however, the complex Cu-Zincon is only partially reversible. The free Zincon, and the complexes Zn-Zincon and Cu-Zincon, does not partition into organic solvents, does not permeate liposome membrane, and neither does it interact with biological membranes. All these characteristics make the metallochromic Zincon useful in biological systems.     

Electric conductivity changes in salt-free solutions in connection with the formation of polyriboadenylic and polyribouridylic acid complexes

Conductometric and spectrophotometric investigations of concentrated salt-free solutions of poly(A) -- poly(U) demonstrated the 1:1 complex formation. It was accomplished by the increase of solution conductivity in contrast to the situation when DNA redenaturation takes place.     

Functional characteristics of normal and pathologic erythrocytes

Polarographic titration in a closed cell of blood samples and concentrated Hb solutions was used to study the regulation of the release of oxygen by the red cell. The effect of temperature, polyethylenglycol and metabolites normally found in the erythrocyte was determined by spectrophotometric and polarographic methods. Some pecularities of O2-transport and release in beta-thalassemia, functional hypoxia and Cooley-disease are described.     

Luxol Fast Blue Mbs: A Stain for Phase Contrast Microscopy

A staining method to increase the contrast of sectioned material for phase contrast microscopy is described. Two stock solutions of the stain are required. The first is made by dissolving 2 gm of luxol fast blue MBS in 100 ml of 95% ethanol. The second solution is made up of 4 ml of a 29% aqueous solution of FeCl₃, 95 ml of 95% ethanol, and 1 ml of concentrated HCl. The staining solution is made by mixing equal parts of the two solutions. Sections are deparaffinized and taken to 70% alcohol, stained for 1.5 hr, dehydrated, cleared and covered as usual.     

Luxol Fast Blue Mbs: A Stain for Phase Contrast Microscopy

A staining method to increase the contrast of sectioned material for phase contrast microscopy is described. Two stock solutions of the stain are required. The first is made by dissolving 2 gm of luxol fast blue MBS in 100 ml of 95% ethanol. The second solution is made up of 4 ml of a 29% aqueous solution of FeCl₃, 95 ml of 95% ethanol, and 1 ml of concentrated HCl. The staining solution is made by mixing equal parts of the two solutions. Sections are deparaffinized and taken to 70% alcohol, stained for 1.5 hr, dehydrated, cleared and covered as usual.     

The ionic composition of the contractile vacuole fluid of Paramecium mirrors ion transport across the plasma membrane

In vivo K+, Na+, Ca2+, Cl- and H+ activities in the cytosol and the contractile vacuole fluid, the overall cytosolic osmolarity, the fluid segregation rate per contractile vacuole and the membrane potential of the contractile vacuole complex of Paramecium multimicronucleatum were determined in cells adapted to 24 or 124 mosm l(-1) solutions containing as the monovalent cation(s): 1) 2 mmol l(-1) K+; 2) 2 mmol l(-1) Na+; 3) 1 mmol l(-1) K+ plus 1 mmol l(-1) Na+; or 4) 2 mmol l(-1) choline. In cells adapted to a given external osmolarity i) the fluid segregation rate was the same if adapted to either K+ or Na+, twice as high when adapted to solutions containing both K+ and Na+, and reduced by 50% or more in solutions containing only choline, ii) the fluid of the contractile vacuole was always hypertonic to the cytosol while the sum of the ionic activities measured in the fluid of the contractile vacuole was the same in cells adapted to either K+ or Na+, at least 25% higher in cells adapted to solutions containing both K+ and Na+, and was reduced by 55% or more in solutions containing only choline, iii) the cytosolic osmolarity was the same in cells adapted to K+ alone, to Na+ alone or to both K+ and Na+, whereas it was significantly lower in cells adapted to choline. At a given external osmolarity, a positive relationship between the osmotic gradient across the membrane of the contractile vacuole complex and the fluid segregation rate was observed. We conclude that both the plasma membrane and the membrane of the contractile vacuole complex play roles in fluid segregation. The presence of external Na+ moderated K+ uptake and caused the Ca2+ activity in the contractile vacuole fluid to rise dramatically. Thus, Ca2+ can be eliminated through the contractile vacuole complex when Na+ is present externally. The membrane potential of the contractile vacuole complex remained essentially the same regardless of the external ionic conditions and the ionic composition of the fluid of the contractile vacuole. Notwithstanding the large number of V-ATPases in the membrane of the decorated spongiome, the fluid of the contractile vacuole was found to be only mildly acidic, pH 6.4.     

Purification and characterization of trehalose-6-phosphate synthase from Saccharomycopsis fibuligera A11

Mutant A11, a mutant of Saccharomycopsis fibuligera Sdu with low acid and neutral trehalase was found to accumulate over 18% (w/w) trehalose from starch in its cells. In this study, trehalose-6-phosphate synthase (Tps1) was purified to homogeneity from this mutant, with a 30-fold increase in the specific enzyme activity, as compared to the concentrated cell-free extract, from initial cells. The molecular mass of the purified enzyme as determined by SDS-PAGE was 66 kDa. The optimum pH and temperature of the purified enzyme were 6.6 and 37 degrees C, respectively. The enzyme was activated by Ca2+, K+ and Mg2+, with K+ showing the highest activation at 35 mM. On the other hand, Mn2+, Cu2+, Fe3+, Hg2+ and Co2+ inhibited the enzyme. The enzyme was also strongly inhibited by protease inhibitors such as iodoacetic acid, EDTA and PMSF.     

Ordered association of tobacco mosaic virus in the presence of divalent metal ions

The formation of ordered aggregates of tobacco mosaic virus (TMV) in the presence of divalent metal ions has been studied in concentrated (1-25 mg/ml) solutions of the virus. The divalent metal cations Cd2+, Zn2+, Pb2+, Cu2+, and Ni2+ have been found to promote TMV precipitation from solution at a critical concentration Ccrit, which for a given metal depends on the pH and the ionic strength of the solution, but is largely independent of the virus concentration. The TMV precipitate behaves as a nematic liquid crystal and on drying at a glass surface produces highly ordered, optically birefringent films. However, precipitation is not observed with alkali-earth metals such as Ca2+ and Mg2+. The experimental data suggest that, apart from two 'internal' metal-binding sites in each TMV subunit, the virus contains metal-binding sites of a lower affinity which promote cross-linking of TMV rods via metal bridges. The latter seem to be responsible for the precipitation of TMV in the presence of divalent cations at neutral pH. We propose that the metal-induced cross-linking may be the predominant mechanism to account for the limited solubility of a variety of proteins in solution containing metal cations with valence 2 and higher.     

Comparative study of various methods for the extraction of free creatine and phosphocreatine from mouse skeletal muscle

The efficacy of various media regarding the extraction of free creatine and phosphocreatine of mouse skeletal muscle was evaluated. In anesthetized animals tissue was quick-frozen in situ and removed by means of a modified Rongeur forceps cooled in liquid N₂. Homogenization of muscle tissue in 1 m EDTA in 50% (v/v) ethanol at −20°C, which was gradually diluted with ice-cold 0.4 perchloric acid to a final concentration of 0.3 perchloric acid in 12.5% ethanol proved to be the most suitable procedure regarding rapid handling of tissue samples, recovery of total creatine, and the ratio of phosphocreatine to total creatine. Phosphocreatine values as high as 78% of total creatine of skeletal muscle were thus obtained. Extraction of free creatine and phosphocreatine with concentrated ethanolic solutions (50–80%, v/v) was found to be incomplete apparently due to irreversible binding of creatine and phosphocreatine to protein precipitates.     

Electron paramagnetic resonance of copper ion and manganese ion complexes with the ionophore A23187.

The binding of Cu2+ and Mn2+ to the ionophore A23187 in chloroform, 90% ethanol, and sonicated phospholipid dispersions in aqueous mediums has been investigated with electron paramagnetic resonance (epr). The spectra indicated axial symmetry for the Cu2+ complexes and distorted octahedral for the Mn2+ complexes. The coordination between metal ion and its ligands is predominantly ionic in character. The stoichiometry, at the concentrations employed, was found to be 1:2 M2+/ionophore except in 90% ethanol where evidence existed for the 1:1 Cu-A23187 complex, as well. Through competition with Mn2+, the sequence of relative affinities in 90% ethanol was measured to be: Mn2+ greater than La3+ greater than Cu2+ greater than Ca2+ greater than Mg2+ greater than Sr2+. The K A of Mn-A23187 binding is greater than 10 10 M-2. In phospholipid dispersions the spectral characteristics of the Cu complex, particularly g, were observed to be a sensitive function of the hydrocarbon chain mobility. This allowed a calculation of the rotational correlation time of the complex to be made. In sonicated dipalmitoyllecithin was computed to be 10-9 sec, reflecting a local viscosity similar to that sensed by the nitroxide spin-label 2,2,6,6-tetramethylpiperidin-1-oxyl. In a (1:1) lecithin-cholesterol dispersion the complex was significantly more immobilized.     

State of the Na+, K+-ATPase system of the female rat brain cerebral cortex in the postnatal period during ethanol consumption in pregnancy and lactation

The ontogenetic development of the rat brain cortex Na+, K(+)-ATPase and Mg(2+)-ATPase activities under female ethanol (20% v/v) consumption in the third trimester of gestation or in postpartum period was studied. The weight characteristics (body, whole brain and cortex weight) of viable rats on the first day after birth were not affected critically by prenatal alcohol exposure. It is revealed that the delay of postnatal rat growth 10 days after birth under translactational ethanol consumption is accompanied by reliable decrease of plasma membrane Na+, K(+)-ATPase activity in comparison with control animals. The comparable decrease in activities was observed for the ouabain-sensitive and ouabain-resistant Na+, K(+)-ATPase components (isoform species). From the 20th day the differences in enzyme activity were not revealed. Mg(2+)-ATPase increases in postnatal period independent of Na+, K(+)-ATPase activity and it remains insensitive to postnatal maternal alcohol intake. It is suggested, the first ten day period of lactation is critical for ethanol effect on the developmental control of the brain Na+, K(+)-ATPase functional expression and the course of adaptive processes in the rat organism.     

Comparison of yeast strains for batch ethanol fermentation of cheese-whey powder (CWP) solution

AIMS: To test the suitability of cheese whey powder (CWP) solution for ethanol fermentation and to compare performances of different Kluyveromyces marxianus strains for ethanol fermentation from CWP solution. METHODS AND RESULTS: Batch ethanol fermentation of cheese whey (CW), CWP and lactose solutions with the same initial sugar contents were compared by using two different K. marxianus strains and the CWP solution was found to be the most suitable substrate. CWP solution was fermented to ethanol using three different yeast strains and DSMZ-7239 was found to be the most suitable one yielding the highest rate and extent (3.3%, v/v) of ethanol formation. CONCLUSIONS: CWP solution and K. marxianus strain of DSMZ-7239 were found to be more suitable for ethanol fermentation with the highest ethanol yield when compared with the other substrates and the yeast strains tested. SIGNIFICANCE AND IMPACT OF THE STUDY: CWP can be used as a concentrated form of CW for ethanol fermentations with considerable advantages.     

Relationship between myeloperoxidase activity and the ontogenic response of rat gastric mucosa to ethanol.

We have examined the gastric luminal content of Na+, K+, and protein and mucosal levels of myeloperoxidase in rats between the ages of 10 and 60 days in response to luminal instillation of ethanol (20 and 50% w/v). In control animals the appearances of ions and protein and myeloperoxidase activities were low and similar in all age groups. Luminal content of cations and protein increased in response to both 20 and 50% ethanol and were greater in animals older than 20 days when compared with younger rats. However, ethanol treatment resulted in a significant degree of mucosal cellular disruption and erosions in both young and mature rats. Myeloperoxidase activities in response to ethanol were not greater than control until animals were older than 20 days. Treatment of rats aged 10-60 days with intraperitoneal glycogen (1%) resulted in peritoneal granulocyte infiltration. The concentration of peritoneal cells increased as animals aged. With the exception of day 15, the myeloperoxidase content of the peritoneal leukocytes did not vary significantly at other ages examined. These data suggest that (1) mucosal efflux of Na+, K+, and protein in response to luminal ethanol increase as rats age from 10 to 60 days; (2) the ontogenic development of ethanol-induced cation and protein appearance parallel the increase in myeloperoxidase activity in the gastric mucosa; and (3) the increase in mucosal myeloperoxidase activity in response to ethanol likely reflects increased granulocyte infiltration as rats age.     

Ethanol increases calcium permeability of heavy sarcoplasmic reticulum of skeletal muscle

The effects of ethanol on both Ca2+ pump activity and Ca2+-induced Ca2+ release in sarcoplasmic reticulum (SR) of rabbit skeletal muscle were studied. In concentrations of 0.1-1.0%, ethanol conspicuously enhanced Ca2+ release from the heavy fraction of SR, whereas a much smaller effect was noted in the light fraction. When Ca2+-induced Ca2+ release was inhibited by 10 mM Mg2+, the Ca2+ pump activities of both the heavy and light SR were the same; the activities were not significantly influenced by 1% ethanol. Ethanol itself did not release Ca2+ from the heavy SR. However, it appeared to render the heavy SR more permeable to Ca2+, thereby decreasing the amount of storable Ca2+. This action of ethanol may be related to the mechanism of its negative inotropic effect.