Regulation of the shape of unsealed erythrocyte membranes by Mg-ATP and Ca2+

In the presence of MgCl₂ and ATP, the specific viscosity of suspensions of unsealed freezethawed erythrocyte membranes decreased slowly with time at 37 °C. The decrease in viscosity was found to be an index of Mg-ATP-specific induced folding of these membranes. Mg-ATP-dependent shape or viscosity changes were found to be highly temperature dependent and the viscosity of these membranes did not decrease in the presence of 2 mm 5′-adenyl imidodiphosphate and MgCl₂. Cyclic AMP, NaCl, or KCl did not have any effect on the rate of Mg-ATP-induced viscosity decreases. The Mg-ATP-dependent viscosity decreases were inhibited 100% by 1 mm chlorpromazine or 1 mmN-ethylmaleimide. Mg-ATP-dependent viscosity decreases were half-maximally inhibited by 1 μm Ca²⁺ and completely inhibited by 3–5 μm Ca²⁺. Ca²⁺ (5 μm) also inhibited Mg²⁺-dependent phosphorylation 25 to 30% in these membranes. However, if these membranes were preincubated in the absence of Ca²⁺ for greater than 10 min at 37 °C, 5 μm Ca²⁺ no longer inhibited Mg-ATP-dependent viscosity decreases and only inhibited Mg²⁺-dependent phosphorylation 5% in these preincubated membranes. Preincubation of these membranes at 37 °C for 10 min in the absence of Ca²⁺ also resulted in the loss of approximately 40 to 50% of the high-Ca²⁺ affinity Ca + Mg-ATPase activity. The presence of 5 μm Ca²⁺ in the preincubation medium protected against the loss of the inhibitory effect of Ca²⁺ on Mg²⁺-dependent phosphorylation and Mg-ATP-dependent viscosity decreases. The presence of Ca²⁺ in the preincubation medium also protected against the loss of Ca + Mg-ATPase activity in these membranes. It is hypothesized that freeze-thawed erythrocyte membranes contain a Ca²⁺ phosphatase activity which is temperature labile in the absence of Ca²⁺ and that this Ca²⁺ phosphatase activity may be involved in the regulation of shape of these membranes. Also discussed is the possible relationship of this Ca²⁺ phosphatase with Ca + Mg-ATPase activity and the problems inherent in studying Ca²⁺-regulated functions in freeze-thawed erythrocyte membranes.     

Purification and Properties of Phospholipase D from Actinomadura sp. Strain No. 362

An extracellular phospholipase D from Actinomadura sp. Strain No. 362 was purified about 430-fold from the culture filtrate. The purified enzyme preparation was judged to be homogeneous on polyacrylamide gel electrophoresis. The molecular weight and isoelectric point of the enzyme were estimated to be about 50,000—60,000 and 6.4, respectively. The enzyme was most active at pH 5.5 and 50°C in the presence of Triton X-100, but showed the highest activity at pH 7.0 and 60 — 70°C in its absence. The enzyme was stable up to 30°C at pH 7.2 and also stable in the pH range of 4.0 to 8.0 on 2 hr incubation at 25°C. With regard to substrate specificity, this enzyme hydrolysed lecithin best among the phospholipids tested. It was activated by Fe^{3 +}, Al³⁺, Mn^{2 +}, Ca^{2 +}, diethyl ether, sodium deoxycholate and Triton X-100, but was inhibited by cetyl pyridinium chloride and dodecylsulfate.     

Behaviour of adsorbed myosin at the oil-water interface

Interfacial tensions and surface shear viscosities are reported at 7 and 25°C for adsorbed films of myosin at the n-tetradecanebuffer interface (bulk protein concentration 10⁻³ wt%, pH 6.5, 0.5 m KCl). Surface behaviour similar to that of other proteins is observed at 25°C, but unique surface behaviour is observed at 8°C. An unusual feature is a surface viscosity which increases with temperature-particularly in young films.     

The effect of soluble alginate and calcium on β-galactosidase activity produced by the thermotolerant, ethanol-producing yeast strain Kluyveromyces marxianus imb3

Since it has previously been demonstrated that ethanol production by the thermotolerant yeast strain, Kluyveromyces marxianus IMB3 is more efficient in calcium alginate-based immobilization systems during growth on lactose-containing media, it was decided to examine the separate effects of soluble alginate and free calcium on the β-galactosidase activity produced by that organism. It was found that the presence of Ca²⁺ significantly increased the thermal stability of the activity at 45?°C, although the pH?and temperature optima remained the same in the presence and absence of that cation. It was also found that the presence of 2% (w/v) sodium alginate (soluble) had a very limited positive effect on the thermal stability of the enzyme at 45?°C, although it was found that activity was very significantly stimulated at that temperature. The activity was found to have an enhanced thermal stability at 30?°C in the presence of sodium alginate. The presence of sodium alginate in assay mixtures had no significant effect on the Km of the activity for the substrate o-nitrophenyl-β-D-galactoside. The results observed in the presence of either free calcium or soluble alginate may at least partially explain enhanced ethanol production by this microorganism in alginate-based immobilization systems.     

Thin-blooded Antarctic fishes: a rheological comparison of the haemoglobin-free icefishes Chionodraco kathleenae and Cryodraco antarcticus with a red-blooded nototheniid, Pagothenia bernacchii

The icefishes (family Channichthyidae) comprise a unique group of teleost fishes endemic to Antarctic and sub-antarctic seas. All members of the family totally lack haemoglobin. Haematological parameters and viscosity were determined for blood from 11 specimens of two channichthyid species (Chionodraco kathleenae Regan, 1914; Cryodraco antarcticus Dollo, 1900), and 14 specimens of a red-blood Antarctic nototheniid species (Pagothenia bernacchii (Boulenger, 1902)), captured near the Italian research station at Terra Nova Bay, Ross Sea, Antarctica. Channichthyid blood contained only a small number of non-pigmented cells (10 000-40 000 cells μI^?1, depending on species) in contrast to nototheniid blood (360 000-450 000 cells μI^?1 in unstressed specimens). Blood viscosity was measured by cone plate viscometry over a range of shear rates (11.3-450s ^?1), at six temperatures between – 1.8°C and + 15°C. At the ambient Antarctic seawater temperature of – 1.8° C, and at low shear rate (22.5 s^?1), the viscosity of channichthyid blood was relatively low (3.99 ± 0.40 cP) compared with blood taken from unstressed P. bernacchii, which was about 25% more viscous (4.91 ± 0.59 cP). The viscosity of channichthyid blood was almost independent of shear rate, approximating an ideal Newtonian fluid, while the viscosity of nototheniid blood was much more dependent upon both shear rate and temperature, increasing sharply at low shear rates and low temperatures. Viscosity of nototheniid blood varied with haematocrit, which was in turn strongly influenced by stress. Blood samples taken from P. bernacchii under moderate stress induced by handling during acute caudal venepuncture had haematocrit values in the range 15–20% and viscosities of 8-l0cP, while undisturbed specimens sampled through a venous cannula yielded haematocrits of 8–10%. The viscosity of nototheniid plasma did not differ significantly from that of channichthyid whole blood or channichthyid plasma. The higher viscosity of nototheniid blood is attributable to cell content, and in stressed specimens possibly also to adrenergic swelling of erythrocytes. The absence of erythrocytes in channichthyid blood avoids the great increases in viscosity which are induced in corpusculate blood by sub-zero seawater temperatures.     

Optical properties of an outer membrane lipoprotein from Escherichia coli

The infrared spectrum of a structural lipoprotein from the Escherichia coli outer membrane indicated the lipoprotein had and α-helical conformation but no sign for the existence of β-structures. From circular dichroism spectra of the lipoprotein, the α-helical content of the protein was found to be as high as 88% in 0.01–0.03% sodium dodecyl sulfate in the presence of 10^?5 M Mg²⁺ at pH 7.1 and 23° C. When sodium dodecyl sulfate concentration increased higher than 0.1%, the α-helical content of the lipoprotein decreased to about 57%. Divalent cations, such as Mg²⁺ and Mn²⁺, were found to increase the helical content of the lipoprotein. The high α-helical content of the lipoprotein was observed in a wide range of temperatures (23 to 55° C). The significance of the high α-helical content of the lipoprotein is discussed in light of the three-dimensional molecular models of the lipoprotein proposed previously.     

Studies on (Na+ + K+-activated ATPase.: XXXVIII. A 100 000 molecular weight protein as the low-energy phosphorylated intermediate of the enzyme

Phosphorylation of NaI-treated bovine brain cortex microsomes by inorganic phosphate in the presence of Mg²⁺ and ouabain has been studied at 0°C (pH 7.4) and 20°C (pH 7.0). Nearly maximal (90%) and half-maximal phosphorylation are achieved at 20°C within 2 min with 50–155 and 5.6–17 μM ^{3 2}P_i, respectively, and at 0°C within 75 s with 300–600 and 33–66 μM ^{3 2}P_i, respectively. Maximal phosphorylation yields 146 pmol ^{3 2}P · mg⁻¹ protein. Without ouabain (20°C, pH 7.0) less than 25% of the incorporation observed in the presence of ouabain is reached.Preincubation of the native microsomes with Mg²⁺ and K⁺, in order to decompose possibly present high-energy phosphoryl-bonds prior to ouabain treatment, does not affect the maximal phosphate incorporation. This indicates that the inorganic phosphate incorporation is not due to an exchange with high-energy phosphoryl-bonds, which might have been preserved in the microsomal preparations.Phosphorylation of the native microsomes by ATP in the presence of Mg²⁺ and Na⁺ reaches 90 and 50% maximal levels within 15–30 s at 0°C and pH 7.4 at concentrations of [γ-^{3 2}P] ATP of 5–32 and 0.5–3.5 μM, respectively. The maximal phosphorylation level is 149 pmol ³²P · mg⁻¹ protein, equal to that of ouabain-treated microsomes phosphorylated by inorganic phosphate. Both inorganic phosphate and ATP phosphorylate one site per active enzyme subunit of 135 000 molecular weight.From the equilibrium constants for the phosphorylation of ouabain-treated microsomes by inorganic phosphate at 0°C and 20°C standard free-energy changes of −5.4 and −6.8 kcal/mol, respectively, are calculated. These values yield a standard enthalpy change of 14 kcal/mol and an entropy change of 70 cal/mol · ^oK. this charactrizes the reaction as a process driven by an entropy change. p ]The intermediate formed by phosphorylation with p_i has maximal stability at acidic pH, as is the case for the intermediate formed with ATP. solubilization in sodium dodecyl sulfate stabilizes the phosphoryl-bond in the pH range 0f 4–7. The non-solubilized preparation has optimal stability at ph 2–4, the level of which is equal to that of detergent-solubilized intermediate.Sodium dodecyl gel electrophoreses of the microsomes at pH 3, the following incorporation of ³²P_i yields 11 protein bands, only one of which (mol. wt 100 000-106 000) carries the radioactive label. This protein has the same molecule weight as the protein, which is phosphorylated by ATP in the presence of Mg²⁺ and Na⁺.     

Histamine H-1 receptors on rat peritoneal mast cells

In order to characterize the receptor subtype involved in histamine stimulation of increased cyclic AMP levels in rat mast cells with consequent impairment of anaphylactically induced mediator release, the binding of the H-1 receptor antagonist [³H] pyrilamine to mast cells was examined. Pyrilamine bound rapidly, in a saturable and reversible fashion, and with increased binding at 4°C as compared with 21°C and 37°C. [³H] Pyrilamine binding was displaced by H-1 antagonists (tripelnnamine > yrilamine ≧ iphenhydramine) > histamine > the H-2 antagonist, cimetidine. H-1 agonists displaced pyrilamine binding less efficiently than histamine but better than H-2 agonists. Rat mast cells have a single homogeneous population of low affinity (K_D = 222 ± 33 nM) H-1 receptors with a Bmax of 9.7 ± 2.3 pm/10⁶ mast cells and 5.4 ± 0.92 × 10⁶ binding sites per mast cell. Thus, the mast cell has an H-1 type histamine receptor which is probably involved in histamine-induced cyclic AMP increases.     

Statistical evaluation and modeling of cheap substrate-based cultivation medium of Chlorella vulgaris to enhance microalgae lipid as new potential feedstock for biolubricant

Chlorella vulgaris (C. vulgaris) microalga was investigated as a new potential feedstock for the production of biodegradable lubricant. In order to enhance microalgae lipid for biolubricant production, mixotrophic growth of C. vulgaris was optimized using statistical analysis of Plackett–Burman (P-B) and response surface methodology (RSM). A cheap substrate-based medium of molasses and corn steep liquor (CSL) was used instead of expensive mineral salts to reduce the total cost of microalgae production. The effects of molasses and CSL concentration (cheap substrates) and light intensity on the growth of microalgae and their lipid content were analyzed and modeled. Designed models by RSM showed good compatibility with a 95% confidence level when compared to the cultivation system. According to the models, optimal cultivation conditions were obtained with biomass productivity of 0.123 g L^?1 day^?1 and lipid dry weight of 0.64 g L^?1 as 35% of dry weight of C. vulgaris. The extracted microalgae lipid presented useful fatty acid for biolubricant production with viscosities of 42.00 cSt at 40°C and 8.500 cSt at 100°C, viscosity index of 185, flash point of 185°C, and pour point of ?6°C. These properties showed that microalgae lipid could be used as potential feedstock for biolubricant production.     

Poly(rA) • Poly(rU) with Ni2+ Ions at Different Temperatures: Infrared Absorption and Vibrational Circular Dichroism Spectroscopy

Abstract

Phase transitions were studied of the sodium salt of poly(rA) ?poly(rU) induced by elevated temperature without Ni²⁺ and with Ni²⁺ in 0.07 M concentration in D₂O (～0.4 [Ni]/[P]). The temperature was varied from 20° C to 90° C. The double-stranded conformation of poly(rA)?poly(rU) was observed at room temperature (20° C—23° C) with and without Ni²⁺ ions. In the absence of Ni²⁺ ions, partial double- to triple-strand transition of poly(rA) ?poly(rU) occurred at 58° C, whereas only single-stranded molecules existed at 70° C. While poly(rU) did not display significant helical structure, poly(rA) still maintained some helicity at this temperature. Ni²⁺ ions significantly stabilized the triple-helical structure. The temperature range of the stable triple-helix was between 45° C and 70° C with maximum stability around 53° C. Triple-to single-stranded transition of poly(rA) ?poly(rU) occurred around 72° C with loss of base stacking in single-stranded molecules. Stacked or aggregated structures of poly(rA) formed around 86° C. Hysteresis took place in the presence of Ni²⁺ during the reverse transition from the triple-stranded to the double-stranded form upon cooling. Reverse Hoogsteen type of hydrogen-bonding of the third strand in the triplex was suggested to be the most probable model for the triple-helical structure. VCD spectroscopy demonstrated significant advantages over infrared absorption or the related electronic CD spectroscopy.     

Cation-dependent light-induced structural changes in visual receptor membranes

A stearamide spin probe was used to study the light-induced structural changes in Rod Outer Segment Membranes in the presence of sodium and calcium ions. The correlation time (τ_C) for the reorientation of the probe was calculated in the dark and light. In the presence of sodium ions, τ_C = 3.3 × 10^?9 sec in the dark, and 2.7 × 10^?9sec in the light while the opposite was noticed in the presence of calcium ions, τ_C = 2.9 × 10^?9 sec in the dark and 3.6 × 10^?9 sec in the light. The correlation times for reorientation of the probe were also calculated in aqueous glycerol solutions of varying viscosities at 20°C. Comparison of the values of τ_C (dark and light) suggests a change in local mobility in the ROS corresponding to a macroscopic viscosity difference of approximately 150 cp. The significance of calcium ion interaction with negatively charged groups and the formation of a Schiff base is emphasized.     

Investigation of DNA structural changes by infrared spectroscopy

DNA-oriented samples of various origins were studied under different conditions of humidiity and sodium chloride content by means of infrared spectroscopy. (1) Oriented DNA (M. Lysodeikticus, E. coli, calf thymus and salmon sperm) films at 3–4% sodium chloride yield polarized spectra which show drastic changes at relative humidities (r.h.) between 94% and 0% indicative of conformational changes: B form → a form → disordered form The measurements of the infrared dichroism at frequencies of about 1230 cm^?1 and at about 1090 cm^?1 allow one to determine the orientation of the phosphate group, whereas the measurements at 1710 cm^?1 characterize the base orientation. At humidities higher than 90% r.h. (B form) the bisector of OPO forms an angle of 70° relative to the helix axis, whereas at lower humidities, between 75% and 50% r.h. (A form) a rotation to about 45° is observed. Simultaneously, the 0—0 line of phosphate group changes its orientation from 55° to 65° to the helix when B → A transition takes place. The results are in general agreement with that of X-ray diffraction and allow one to determine the orientation of the phosphate group with greater precision. (2) The B–A conformational change is not observed for satellite DNA, isolated from Cancer pagurus, of which the guanine + cytosine content is below 5%. As a function of decreasing humidities, one observes the transition: B form → disordered form A diagram of conformational changes of DNA's as a function of base composition and of r.h., suggests that B–A transition will occur for DNA of relatively higher G + C content, whereas for high (A + T) content, base sequence may be of importance. The B–A transition is prevented in DNA at a relatively high or very low sodium chloride content.     

Discrimination by temperature of opiate agonist and antagonist receptor binding.

Variations in incubation temperature can markedly differentiate opiate receptor binding of agonists and antagonists. In the presence of sodium increasing incubation temperatures from 0° to 30° reduces receptor binding of ³H-naloxone by 50% while tripling the binding of the agonist ³H-dihydromorphine. Lowering incubation temperature from 25° to 0° reduces the potency of morphine in inhibiting ³H-naloxone binding by 9-fold while not affecting the potency of the antagonist nalorphine. At temperatures of 25° and higher the number of binding sites for opiate antagonists is increased by sodium and the number of sites for agonists is decreased by sodium with no changes in affinity. By contrast, in the presence of sodium lowering of incubation temperature to 0° increases opiate receptor binding of the antagonist naloxone by enhancing its affinity for binding sites even though the total number of binding sites are not changed.     

Biochemical properties of the alkaline lipase of Bacillus flexus XJU-1 and its detergent compatibility

The possibility of using Bacillus flexus XJU-1 lipase in detergent preparations was studied. The enzyme was monomeric protein as confirmed by liquid chromatography-mass spectrometry and its molecular weight was 15.95 kDa. The lipase showed optimum activity at pH 10.0 and was 100% stable for 24 h at pH 10.0 and 11.0. It exhibited maximum activity at 70°C and retained more than 70% of the initial activity at 60, 70 and 80°C for 24 h. The activity was stimulated by Ca²⁺, Ba²⁺, Mg²⁺ and Co²⁺, whereas 50% of the initial activity was lost with Fe³⁺ and Hg²⁺. The activity was inhibited by 10 mM N-bromosuccinimide and tosyl-L-lysylchloromethylketone, while N-ethylmaleimide, phenylmethylsulphonylfluoride and urea did not show any effect. The enzyme significantly hydrolysed olive, cottonseed, sunflower, groundnut, and gingelly oils. With p-nitrophenyl palmitate, V_max and K_m were 62.5 U/mL and 2.25 mM, respectively. The lipase maintained its stability in Tween-80, Triton-100 and H₂O₂ at 1%, but an activation of 10% and a reduction of 15% in relative activity were observed with NaClO and sodium dodecyl sulphate, respectively. The enzyme retained maximum storage stability for 20 days at ?20, 4 and 30°C. In the presence of 0.7% (w/v) Ariel, Henko, Super wheel, Tide plus and Rin, a retention of more than 84.90% initial activity was recorded after 24 h at 60°C. The supplementation of the lipase to the detergents improved the olive oil stain removal. These properties suggested the present enzyme as a potential additive for detergent preparations.     

pH-dependent effects of sodium tungstate on the steroid-binding properties of hen oviduct progesterone receptor

Effects of sodium tungstate on the steroid-binding properties of hen oviduct progesterone receptor were examined and were found to be pH-dependent. When freshly prepared hen oviduct cytosol containing progesterone receptor was heated at 37°C for 20 min, its ability to bind [³H]progesterone decreased to 20% level of unheated samples. At pH 7, presence of 2–3 mM tungstate during the above incubation period reduced this loss of binding. At higher tungstate concentrations (>5 mM), this stabilizing effect was gradually abolished. Similar results were obtained with preparations that contained [³H]progesterone-receptor complexes; 70–80% of which remained after a 20 min incubation at 37°C in the presence of 2–3 mM tungstate at pH 7. At pH 8, presence of tungstate (1–10 mM) during the 37°C incubation stabilized both the steroid-bound and the unoccupied progesterone receptor in a concentration-dependent manner. The extent of steroid binding by the receptor at 4°C remained unchanged in the presence of up to 10 mM tungstate at both pH 7 and pH 8 assay conditions while presence of 20 mM tungstate lowered this binding capacity. These results indicate that tungstate effects may be mediated via its interaction with the progesterone receptor.     

Uncoupling of Occlusion From ATP Hydrolysis Activity in Sarcoplasmic Reticulum (Ca2++Mg2+)-ATPase

The uncoupling of Ca²⁺ transport from ATP hydrolysis in the sarcoplasmic reticulum (Ca²⁺ + Mg²⁺)-ATPase by trypsin digestion was re-investigated by comparing ATPase activity with the ability of the enzyme to occlude Eu³⁺ (a transport parameter) after various tryptic digests. With this method, re-examination of uncoupling by tryptic digest of the ATPase revealed that TD2 cleavage (Arg-198) had no effect on either occlusion or ATPase activity. Digestion past TD2 in the presence of 5 mM Co²⁺ and at 25°C resulted in the loss of about 70% of the ATPase activity, but no loss of occlusion. Digestion past TD2 in the presence of 5 mM Ca²⁺, 3 mM ATP, and at 25°C resulted in a partially uncoupled enzyme complex which retained about 50% of the ATPase activity, but completely lost the ability to occlude Eu³⁺. Digest past TD2 in the presence of 5 mM Ca²⁺ and 3 mM AMP-PNP. (a non-hydrolyzable ATP analog) at 25°C resulted in no loss of occlusion, thus revealing the absolute requirement of ATP during the digest to eliminate occlusion. From these findings we conclude that uncoupling of Ca²⁺ transport from ATPase activity is possible by tryptic digestion of the (Ca²⁺ + Mg²⁺)-ATPase. Interestingly, only after phosphorylation of the enzyme do the susceptible bond(s) which lead to the loss of occlusion become exposed to trypsin.     

Purification and Characterization of a Thermostable Alkaline Protease from Alkalophilic Thermoactinomyces sp. H8682

Protease secreted into the culture medium by alkalophilic Thermoactinomyces sp. HS682 was purified to an electrophoretically homogeneous state through only two chromatograhies using Butyl-Toyopearl 650M and SP-Toyopearl 650S columns. The purified enzyme has an apparent relative molecular mass of 25, 000 according to gel filtration on a Sephadex G-75 column and SDS-PAGE and an isoelectric point above 11.0.

Its proteolytic activity was inhibited by active-site inhibitors of serine protease, DFP and PMSF, and metal ions, Cu²⁺ and Hg²⁺. The enzyme was stable toward some detergents, sodium perborate, sodium triphosphate, sodium-n-dodecylbenzenesulfonate, and sodium dodecyl sulfate, at a concentration of 0.1% and pH 11.5 and 37°C for 60 min. The optimum pH was pH 11.5–13.0 at 37°C and the optimum temperature was 70°C at pH 11.5. Calcium divalent cation raised the pH and heat stabilities of the enzyme. In the presence of 5 mM CaCl₂, it showed maximum proteolytic activity at 80°C and stability from pH 4–12.5 at 60°C and below 75°C at pH 11.5. The stabilization by Ca²⁺ was observed in secondary conformation deduced from the circular dichroic spectrum of the enzyme. The protease hydrolyzed the ester bond of benzoyl leucine ester well. The amino acid terminal sequence of the enzyme showed high homology with those of Microbiol serine protease, although alanine of the NH₂-terminal amino acid was deleted.     

An organic solvent-tolerant protease from Pseudomonas aeruginosa strain K: Nutritional factors affecting protease production

An organic solvent-tolerant bacterium producing an organic solvent-stable protease was isolated from soil and identified as Pseudomonas aeruginosa strain K. Nutritional requirements for optimized protease production by this strain were investigated. Maximum protease activity was achieved with sorbitol as the sole carbon source, followed by starch and lactose at pH 7.0 and 37 °C. Dextrose, sucrose and glycerol greatly reduced the protease production. The best organic nitrogen source was casamino acid. Tryptone, soytone and yeast extract supported protease production while corn steep liquor and beef extract inhibited the protease activity. Significant protease production was observed with sodium nitrate as a sole nitrogen source however, ammonium nitrate completely inhibit it. More than 62% drop in production occurred in the presence of amino acids. Addition of metal ions such as K⁺, Mg²⁺ and Ca²⁺ maximized the enzyme production.     

Rheology of hyaluronan solutions under extensional flow

The extensional viscosity and the steady shear viscosity of sodium type hyaluronan (NaHA) in water with sodium chloride and/or sucrose and in DMSO solvent were measured. The extensional viscosities for HA in aqueous solution (0.05, 0.1, 0.3 w/v%) were constant at lower extensional rates, and then became strain thinning above a critical extensional rate. However, on adding sodium chloride, the extensional viscosity decreased and became strain thickening at higher extensional rates. Sodium ions shield the electrostatic repulsion between carboxyl residues of HA molecules and constrict the coil dimensions. The strain thickening of HA solution in the presence of sodium chloride at higher extension rates is due to the coil stretching. The addition of sucrose increased the extensional viscosity and shifted the critical extensional rate to lower strain rates. With increasing strain (shear) rates, extensional (shear) viscosities for HA aqueous solutions remained constant up to a critical extension (shear) rate; but they showed no plateau and decreased linearly in DMSO. It is clear that molecular interaction of HA in DMSO is stronger than that in aqueous solution. This should be attributed to the different conformations of HA in DMSO and in aqueous solutions.     

Effect of γ-Irradiation on Development of Lachrymator of Onion

A thermosensitive uracil requiring mutant of Bacillus subtilis Marburg 168 thy trp₂ ts42 was examined as to the colony forming ability at the permissive and nonpermissive temperatures. The viability of the mutant cells decreased rapidly at the restrictive temperature in the modified Woese’s (MW) medium. However, the cells retained viability when sodium succinate or potassium chloride was added to the medium at that temperature although uracil deficiency was unchanged. A little but significant incorporation of adenine-8-¹⁴C into RNA still continued even after the incorporation of N-acetyl-³H-d-glucosamine into acid insoluble fraction of the cells terminated in the MW medium at 48°C. Both incorporations as well as increase of absorbance were slowed down in the presence of sodium succinate at 48°C. This mutant, ts42, was more sensitive to deoxycholate (DOC) than the parent strain. The restoration of colony forming ability after the temperature shift back from 48 to 37°C was suppressed by the addition of DOC to the medium. However, the cell became resistant to DOC when uracil was added to the medium prior to the temperature shift.