Studies on the viscosity behavior of concentrated sodium and potassium halides in aqueous d-mannitol solution

The viscosity behavior of added sodium and potassium halides (in the concentration range 0.125-3m), in aqueous (0.4m) d-mannitol solution, over the temperature range 25–40° has been investigated. It has been found that Moulik's equation holds good for these systems beyond the Einstein region. From the relative viscosity data, the “effective”, rigid molar volume, V_e, and apparent B coefficient have been computed by employing the Breslau-Miller treatment. On the basis of the values of the apparent B coefficient of these electrolytes, the change in viscosity behavior is attributed to structure making/breaking effects in solution.     

Studies on electrolyte-nonelectrolyte interactions: Viscosity behavior of alkali halides aqueous sucrose solutions

The effect of various added electrolytes (sodium and potassium halides of progressively increasing molecular volume) on the viscosity behavior of aqueous sucrose solution (292mM) has been investigated. Data are presented for the concentration range 0.125–3M of the electrolytes over the temperature range of 25 to 40°. The data have been found to satisfy Moulik's equation,(η/η₀)²M +K¹C², for concentrated solutions of electrolytes beyond the Einstein region. The “effective” rigid, molar volume (V_e) and apparent. Jones-Dole B-coefficient (in sucrose solution) have been computed by employing the Breslau-Miller treatment. The change in the viscosity behavior is attributed to structuring effects in solution.     

Hofmeister effects on activity and stability of alkaline phosphatase

We have studied the effects on alkaline phosphatase of adding high concentrations (normally 1.0 M) of simple salts. It is necessary to allow for significant effects of salts on the extinction coefficient of the reaction product, and on the apparent pH of the buffer. Both activity and stability of the enzyme correlate well with the Hofmeister series in terms of the salt's kosmotropic/chaotropic properties, which are assessed by the Jones–Dole viscosity B coefficients (B₊ for cations and B₋ for anions). The catalytic activity or V_max/K_m of the enzyme showed a bell-shaped relationship with the (B₋ − B₊) values of the salts present, being optimal with salts (such as NaCl, KCl, and KNO₃) where the anion and cation have similar kosmotropic/chaotropic properties. This effect is believed to be enzyme-specific and relates to the impact of both cations and anions on the enzyme's surface pH, active site, and catalytic mechanism. Anions play a more predominant role than cations in affecting enzyme stability. The rate of irreversible thermal inactivation is strongly reduced by addition of kosmotropic anions like SO₄²⁻ (half-life increased from 8 to 580 min at 60 °C). This effect is general and the mechanism probably involves the ability of the ions to affect the water solvation layer around the enzyme molecule and to interact with both the surface and internal structure of the enzyme.     

The effect of temperature upon the combined diffusional and kinetic parameters of tissue respiration.

The parameter of temperature has been introduced into equations which relate respiration rate and the diffusion of oxygen into respiring tissues having different geometries. A general equation has been derived having the form $\text{d = A(C)}{}^{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{exp}\text{(}\text{f}\text{2RT}$), where d is a measure of the depth of tissue which can be oxygenated; C is the external oxygen partial pressure; A is a complex constant which can, however, be evaluated for any given tissue and geometry; and f is given by the relationship f =μ — (ΔH + B), where μ is the Arrhenius coefficient of energy of activation of the respiratory complex within the tissue; ΔH is the differential molar heat of the solution of oxygen in water; and B is the (empirical) temperature coefficient of the viscosity of water. The equation is evaluated for the two situations of sheets and spheres of respiring tissue.     

Effect of mineral salts on apparent viscosity and a ratio of product components in josamycin fermentation

3-O-Deacetyl-josamycin (DeAcJM) was accumulated as a by-product in josamycin fermentation by Streptomyces narbonensis var. josamyceticus. Josamycin accumulation was apparently related to the degree of mycelial growth and the accelerative increase in apparent viscosity of the broth. However, the oxygen transfer coefficient was reduced by an increase in the apparent visensity and had a significant influence on josamycin formation. Josamycin accumulation was found to be inversely proportional to the DeAcJM-ratio, defined as the ratio of DeAcJM to the sum of josamycin and DeAcJM. Several ingredients of the medium and fermentation conditions were observed to alter the DeAcJM-ratio. In particular, the addition of mineral salts acted dramatically to reduce the apparent viscosity and DeAcJM-ratio. The reduction of the apparent viscosity causes improvement in the oxygen transfer coefficient. This brings about a considerable decrease in the DeAcJM-ratio along with a convergence in josamycin while increasing josamycin accumulation.     

Studies on the viscosity behavior of concentrated alkali halides in aqueous maltose solutions

The viscosities of concentrated solutions of sodium and potassium halides (concentration range 0.125 to 3.0m) have been measured in aqueous maltose solution at 25, 30, 35, and 40°. Various equations employed for concentrated solutions of electrolytes have been tested, to ascertain the validity of the relative viscosity data. In order to elucidate the structural behavior of sodium and potassium halides in aqueous maltose solution, the molar volumes ($\text{V}$), ionic B-coefficients, and hydration numbers (n_B) of various ions have been computed. The B₊ and B_? coefficients have been interpreted in terms of solute-solvent interactions. On the basis of the data, it has been found that, in 0.5m maltose solution, the different ions show structure-breaking tendency in the order: I^? > Br^? > Cl^? > K⁺ > Na⁺.     

Construction of Predictive Models to Describe Apparent and Complex Viscosity Values of O/W Model System Meat Emulsions Using Adaptive Neuro – Fuzzy Inference System (ANFIS) and Artificial Neural Networks (ANN)

This paper introduces an adaptive neuro ?C fuzzy inference system (ANFIS) and artificial neural networks (ANN) models to predict the apparent and complex viscosity values of model system meat emulsions. Constructed models were compared with multiple linear regression (MLR) modeling based on their estimation performance. The root mean square error (RMSE), mean absolute error (MAE) and determination coefficient (R ²) statistics were performed to evaluate the accuracy of the models tested. Comparison of the models showed that the ANFIS model performed better than the ANN and MLR models to estimate the apparent and complex viscosity values of the model system meat emulsions. Coefficients of determination (R ²) calculated for estimation performance of ANFIS modeling to predict apparent and complex viscosity of the emulsions were 0.996 and 0.992, respectively. Similar R ² values (0.991 and 0.985) were obtained when estimating the performance of the ANN model. In the present study, use of the constructed ANFIS models can be suggested to effectively predict the apparent and complex viscosity values of model system meat emulsions.     

STUDIES ON THE ANOMALOUS VISCOSITY AND FLOW-BIREFRINGENCE OF PROTEIN SOLUTIONS : II. ON DILUTE SOLUTIONS OF PROTEINS FROM EMBRYONIC AND OTHER TISSUES

1. An extensive investigation has been made of protein particle shape using the methods of flow-birefringence and anomalous viscosity measurement in the coaxial cell. 2. As a result of investigations on a number of proteins, it is concluded that they may be divided into four groups. Group A consists of those which show flow-anomaly both in the bulk phase and in the surface film. These also show flow-birefringence in the bulk phase. Examples: tobacco mosaic disease virus nucleoprotein; myosin. Though corpuscular proteins, they have elongated particles before denaturation. Group B consists of those which show flow-anomaly only (in the first instance) in the surface film, and no flow-birefringence in the bulk phase. They are probably close to spherical in shape in solution, but form elongated particles as they denature in the surface film. After this process has been completed, they may show flow-anomaly also in the bulk phase. Some proteins show flow-anomaly in the surface film immediately it forms, others only show it after a certain time has elapsed for the building up of the film. We designate the former as group B₁ and the latter as group B₂. Group B₁, immediate surface film flow-anomaly. Examples: serum euglobulin, amphibian embryo euglobulin b. Group B₂, slowly appearing surface film flow-anomaly. After the film has once been fully formed and then dispersed by shaking, the solution may have the properties of that of a protein in group B₁; i.e., anomalous flow in the film may occur immediately on testing in the viscosimeter. Examples: avian ovalbumin, amphibian embryo pseudoglobulin. Group C consists of those proteins which show flow-anomaly neither in the bulk phase nor in the surface film, under the conditions used by us. They are probably close to spherical in shape. Examples: insulin, methaemoglobin, amphibian embryo euglobulin c, mucoproteins. 3. The theoretical significance of protein fibre molecules, whether native or formed by denaturation in the living cell, is discussed, especially in relation to experimental morphology and cytology.     

The kinetics of intrinsic factor-vitamin B12 binding

The kinetic behaviour of intrinsic factor-vitamin B₁₂ binding has been examined under varying conditions using an albuminised charcoal separation technique. The overall reaction obeys second order rate laws. The intrinsic factor considered alone obeys first order laws; the velocity of reaction of vitamin B₁₂ is too fast for measurement by the technique described but by deduction obeys first order laws. Rate constants as three temperatures, (k₂ at 25°C=1.56·10⁸·mole^?1·s^?1) the activation energy (E=12.7 kJ·mole^?1) and Arrhenius constant (A=2.7·10¹⁰ 1·mole^?1·s^?1 have been calculated. There is the possibility of diffusion control of the reaction in which case the E and A values are invalid. The effect of pH on the reaction has been studied and the results discussed in relation to the pH studies of other workers whose results show disagreement. Albumin coated charcoal was shown to discriminate between intrinsic factor-vitamin B₁₂ and free vitamn B₁₂ over a wide pH range. The apparent under-estimation of intrinsic factor in dilute solution was shown to be due to adsorption of the intrinsic factor to plastic tubes.     

Cellular determinants of low-shear blood viscosity

Low-shear viscometry is one of the methods commonly used to estimate the degree of red blood cell (RBC) aggregation in various bloods and RBC suspensions. However, it has been previously shown that alterations in RBC morphology and mechanical behavior can affect the low-shear apparent viscosity of RBC suspensions; RBC aggregation is also sensitive to these cellular factors. This study used heat treatment (48°C, 5 min), glutaraldehyde (0.005–0.02%) and hydrogen peroxide (1 mM) to modify cell geometry and deformability. Red blood cell aggregation was assessed via a Myrenne Aggregometer (“M” and “Ml” indexes), RBC suspension viscosity was measured using a Contraves LS-30 viscometer, and RBC shape response to fluid shear stresses (i.e., deformability) was determined by ektacytometry (LORCA system). Our results indicate that low-shear apparent viscosity and related indexes may not always reflect changes of RBC aggregation if cellular properties are altered: for situations where RBC aggregation has been only moderately affected, cellular mechanical factors may be the major determinant of low-shear viscosity. These findings thus imply that in situations which may be associated alterations of RBC geometry and/or deformability, low-shear viscometry should not be the sole measurement technique used to assess RBC aggregation.     

The presence of LHRH-like receptors in Dunning R3327H prostate tumors

Quantitative analyses of LH-RH-like membrane receptors were performed in five tumors from the transplantable Dunning R3372H rat prostatic adenocarcinoma. The binding of D-Trp⁶-LH-RH, an agonist of LH-RH, was observed in all 5 tumors. The antagonist [Ac-Dp-Cl-Phe^1,2,D-Trp³,D-Lys⁶,D-Ala¹⁰]-LH-RH was bound to 4 tumors. The apparent equilibrium dissociation constant (K_d) for D-Trp⁶-LH-RH receptor was from 2.6–3.9 × 10^?10 M. The apparent equilibrium B_max values (maximum number of binding sites) were from 17.2–86.0 fmol/mg membrane protein for D-Trp⁶-LH-RH receptor. The K_d for the antagonist was from 2.4–2.7 × 10^?10 M and the B_max values were from 35.5–66.0 fmol/mg membrane protein. Similar binding studies performed in 6 normal rat prostates showed no binding capacities.     

Mise en évidence et évaluation des “moyennes molécules” de la taille de la vitamine B12 présentes dans les liquides biologiques de sujets normaux et de patients urémiques

Determination of “middle molecules” presenting vitamin B₁₂ molecular size in normal and uremic body fluidsUremic solutes with the molecular size of vitamin B₁₂ are assumed to be toxic. An analytical method is proposed to detect and separate these solutes in body fluids using two combined techniques: gel filtration on Sephadex G-15 and ion-exchange chromatography on DEAE-Sephadex A-25. The vitamin B₁₂ molecular size has been localized by ultrafiltration through membranes with a defined cut-off. Normal and uremic body fluids (urine, plasma, hemodialysis fluid) have been separated into 9 ultraviolet-absorbing peaks (a to i) by high-speed gel filtration. Peaks b and e present the molecular size of vitamin B₁₂, 10–15 Å molecular diameter in pH 7 aqueous solution. Peak b, which correlates with uremic neuropathy, is separated into 6 sub-peaks (b₁ to b₆) by ion-exchange chromatography, sub-peak b_4.2 is the only one to correlate with uremic neuropathy. The coefficient of variation in the integrated area of a single peak is 16%. This method gives the chromatographic profile of the vitamin B₁₂ molecular size content from 500 μl of uremic plasma or 100 μl of normal urine within one hour.     

In Situ Characterization of Splenic Brucella melitensis Reservoir Cells during the Chronic Phase of Infection in Susceptible Mice

Brucella are facultative intracellular Gram-negative coccobacilli that chronically infect humans as well as domestic and wild-type mammals, and cause brucellosis. Alternatively activated macrophages (M2a) induced by IL-4/IL-13 via STAT6 signaling pathways have been frequently described as a favorable niche for long-term persistence of intracellular pathogens. Based on the observation that M2a-like macrophages are induced in the spleen during the chronic phase of B. abortus infection in mice and are strongly infected in vitro, it has been suggested that M2a macrophages could be a potential in vivo niche for Brucella. In order to test this hypothesis, we used a model in which infected cells can be observed directly in situ and where the differentiation of M2a macrophages is favored by the absence of an IL-12-dependent Th1 response. We performed an in situ analysis by fluorescent microscopy of the phenotype of B. melitensis infected spleen cells from intranasally infected IL-12p40^-/- BALB/c mice and the impact of STAT6 deficiency on this phenotype. Most of the infected spleen cells contained high levels of lipids and expressed CD11c and CD205 dendritic cell markers and Arginase1, but were negative for the M2a markers Fizz1 or CD301. Furthermore, STAT6 deficiency had no effect on bacterial growth or the reservoir cell phenotype in vivo, leading us to conclude that, in our model, the infected cells were not Th2-induced M2a macrophages. This characterization of B. melitensis reservoir cells could provide a better understanding of Brucella persistence in the host and lead to the design of more efficient therapeutic strategies.     

Chain conformation and rheological behavior of an extracellular heteropolysaccharide Erwinia gum in aqueous solution

The chain conformation of a heteropolysaccharide Erwinia gum (EG) consisting of Glc, Gal, Fuc, and GlcA in aqueous solution was investigated by using viscometry and static and dynamic light scattering. The Huggins constants k′ ranging from 0.31 to 0.35, and the larger second virial coefficient A₂ of the order of 10⁻⁴ and even 10⁻³ mol g⁻² cm³ for EG fractions having different molecular weights in 0.03 M NaCl aqueous solution at 25 °C, suggested that 0.03 M NaCl aqueous solution is a good solvent for EG polysaccharide. Smidsrød’s ‘B-value’ characterizing chain stiffness was estimated to be 0.028-0.045 for EG fractions indicating that the backbone of EG polysaccharide is semi-stiff having similar stiffness to the semi-stiff Alginate and CMC. The hydrodynamic factor ρ (1.69-1.89), Flory-Fox factor Φ, and the product of ρΦ/N_A (0.16-0.22) also confirmed the semi-stiffness of EG polysaccharide chains. Compared with general flexible polymers, the first remarkable shear-thinning and then Newtonian flowing behaviors in steady shear tests for EG polysaccharides were ascribed to the alignment of extended semi-stiff chains on shearing. The dynamic oscillatory shear experiments indicated that addition of certain amount of NaCl effectively prohibited its gelation in pure water even at high concentration and low temperature for long time, suggesting that 0.03 M NaCl aqueous solution of EG has good stability and ability of antigelation, and thus is a promising additive in food field.     

Agrobacterium tumefaciens-mediated genetic transformation of mungbean (Vigna radiata L. Wilczek) — a recalcitrant grain legume

Agrobacterium-mediated transformation of Vigna radiata L. Wilczek has been achieved. Hypocotyl and primary leaves excised from 2-day-old in-vitro grown seedlings produced transgenic calli on B₅ basal medium supplemented with 5×10⁻⁶ M BAP, 2.5×10⁻⁶ M each of 2,4-D and NAA and 50 mg l⁻¹ kanamycin after co-cultivation with Agrobacterium tumefaciens strains, LBA4404 (pTOK233), EHA105 (pBin9GusInt) and C58C1 (pIG121Hm) all containing β-glucuronidase (gusA) and neomycin phosphotransferase II (nptII) marker genes. Transformed calli were found resistant to kanamycin up to 1000 mg^.l⁻¹. Gene expression of kanamycin resistance (nptII) and gusA in transformed calli was demonstrated by nptII assay and GUS histochemical analysis, respectively. Stable integration of T-DNA into the genome of transformed calli of mungbean was confirmed by Southern blot analysis. Transgenic calli could not regenerate shoots on B₅ or B₅ containing different cytokinins or auxins alone or in combination. However, for the first time, transformed green shoots showing strong GUS activity were regenerated directly from cotyledonary node explants cultured after co-cultivation with LBA4404 (pTOK233) on B₅ medium containing 6-benzylaminopurine (5×10⁻⁷ M) and 75 mg l⁻¹ kanamycin. The putative transformed shoots were rooted on B₅+indole-3-butyric acid (5×10⁻⁶ M) within 10–14 days and resulted plantlets subsequently developed flowers and pods with viable seeds in vitro after 20 days of root induction. The stamens, pollen grains and T₀ seeds showed GUS activity. Molecular analysis of putative transformed plants revealed the integration and expression of transgenes in T₀ plants and their seeds.     

Ent-Kaurene Biosynthesis in Extracts of Helianthus annuus L. Seedlings

Kaurene synthetase B activity (conversion of copalyl pyrophosphate to ent-kaurene) is readily detectable in crude cell-free extracts of 3- to 4-day old dark-grown sunflower (Helianthus annuus cv. Mammoth) seedlings, whereas little or no kaurene synthetase AB activity (conversion of geranylgeranyl pyrophosphate to ent-kaurene) can be found in these extracts under comparable assay conditions. A low amount of AB activity is evident only if an extensively dialyzed extract is used in low concentrations as the enzyme source. One factor which may contribute to the low apparent levels of AB activity is the presence of inhibitory factors in the crude sunflower extract since these extracts can be shown to act as a potent inhibitor of Marah macrocarpus endosperm kaurene synthetase AB activity. Heat treatment (100°C) or dialysis of the sunflower extract reduces the amount of its inhibitory activity. Also, it was observed that low concentrations of extensively dialyzed sunflower extracts act to stimulate M. macrocarpus AB activity. There is no evidence for the presence of an inhibitory factor for M. macrocarpus kaurene synthetase B activity in sunflower extracts. However, there does appear to be present in the crude preparation of sunflower extract a dialyzable factor(s) that impedes its own B activity. There is little information to date on the nature of these inhibitory and stimulatory factors for kaurene synthetase activity or their possible roles in physiological regulation. The possible presence of such factors should be considered, however, when attempting to evaluate kaurene synthetase activities in extracts of vegetative plants.     

Protein Mobility in the Cytoplasm of Escherichia coli

The rate of protein diffusion in bacterial cytoplasm may constrain a variety of cellular functions and limit the rates of many biochemical reactions in vivo. In this paper, we report noninvasive measurements of the apparent diffusion coefficient of green fluorescent protein (GFP) in the cytoplasm of Escherichia coli. These measurements were made in two ways: by photobleaching of GFP fluorescence and by photoactivation of a red-emitting fluorescent state of GFP (M. B. Elowitz, M. G. Surette, P. E. Wolf, J. Stock, and S. Leibler, Curr. Biol. 7:809–812, 1997). The apparent diffusion coefficient, D_a, of GFP in E. coli DH5α was found to be 7.7 ± 2.5 μm²/s. A 72-kDa fusion protein composed of GFP and a cytoplasmically localized maltose binding protein domain moves more slowly, with D_a of 2.5 ± 0.6 μm²/s. In addition, GFP mobility can depend strongly on at least two factors: first, D_a is reduced to 3.6 ± 0.7 μm²/s at high levels of GFP expression; second, the addition to GFP of a small tag consisting of six histidine residues reduces D_a to 4.0 ± 2.0 μm²/s. Thus, a single effective cytoplasmic viscosity cannot explain all values of D_a reported here. These measurements have implications for the understanding of intracellular biochemical networks.     

Evaluation of a Brucella melitensis mutant deficient in O-polysaccharide export system ATP-binding protein as a rough vaccine candidate

Rough Brucella mutants have been sought as vaccine candidates that do not interfere with the conventional serological diagnosis of brucellosis. In this study, a rough mutant of Brucella melitensis was generated by the disruption of the wzt gene, which encodes the O-polysaccharide (O-PS) export system ATP-binding protein. In vivo, the mutant 16MΔwzt was attenuated and conferred a level of protection against B. melitensis 16M challenge similar to that conferred by the vaccine strain B. melitensis M5 in mice. In pregnant sheep, the mutant 16MΔwzt did not induce abortion. In vitro, 16MΔwzt was more susceptible to polymyxin B and complement-mediated killing than B. melitensis 16M was. Most importantly, although 16MΔwzt had a rough phenotype, it was able to synthesize O-PS and did not induce detectable specific antibodies in sheep. These results suggested that 16MΔwzt deserved to further systematic evaluation as a vaccine for target animal hosts due to its promising features.     

Selection by Anion-Exchange Chromatography of Exopolysaccharide Mutants of the Cyanobacterium Synechocystis Strain PCC 6803

The degree of retention of whole cells of Synechocystis strain PCC 6803 on DEAE-cellulose columns was shown to depend on their content of exopolysaccharides, which are at least in part responsible for the external negative charge of the cells. This feature was used for the isolation of mutants modified in the apparent viscosity caused by these macromolecular constituents. When a wild-type suspension was loaded onto a DE52 column, the cells eluting in the two extreme fractions of a 0 to 5 M NaCl step gradient represented 10⁻⁹ to 10⁻⁷ of the total eluted population. The accuracy of the procedure was established through the analysis of four clones: Suc(0)32 and Suc(0)65 (0 M) and Suc(5)64A and Suc(5)61 (5 M). The decreased viscosity of the exopolymers of the two 0 M clones, which appeared identical, could be related to the production of molecules less charged in uronic acids and more readily liberated from the cells. The two 5 M clones exhibited a lower sedimentation velocity, correlating with either a 60% increase in uronic acid and a doubling of the specific viscosity of the exopolysaccharides [clone Suc(5)64A] or a doubling of the per-cell production of polymers otherwise identical to those from wild-type cells [clone Suc(5)61].     

Sensitivity of Meloidogyne incognita and Rotylenchulus reniformis to Abamectin

Avermectins are macrocyclic lactones produced by Streptomyces avermitilis. Abamectin is a blend of B_1a and B_1b avermectins that is being used as a seed treatment to control plant-parasitic nematodes on cotton and some vegetable crops. No LD₅₀ values, data on nematode recovery following brief exposure, or effects of sublethal concentrations on infectivity of the plant-parasitic nematodes Meloidogyne incognita or Rotylenchulus reniformis are available. Using an assay of nematode mobility, LD₅₀ values of 1.56 μg/ml and 32.9 μg/ml were calculated based on 2 hr exposure for M. incognita and R. reniformis, respectively. There was no recovery of either nematode after exposure for 1 hr. Mortality of M. incognita continued to increase following a 1 hr exposure, whereas R. reniformis mortality remained unchanged at 24 hr after the nematodes were removed from the abamectin solution. Sublethal concentrations of 1.56 to 0.39 μg/ml for M. incognita and 32.9 to 8.2 μg/ml for R. reniformis reduced infectivity of each nematode on tomato roots. The toxicity of abamectin to these nematodes was comparable to that of aldicarb.