Lateral diffusion rates of lipid,water, and a hydrophobic drug in a multilamellar liposome

The lateral diffusion constants of 1-palmitoyl-2-oleoyl-sn-glycero-3 phosphocholine (POPC), water, and ibuprofen were measured in multilamellar liposomes using pulsed field gradient magic-angle spinning (PFG-MAS) (1)H NMR. The analysis of diffusion data obtained in powder samples and a method for liposome curvature correction are presented. At 322 K POPC has a diffusion constant of (8.6 +/- 0.2) x 10(-12) m(2)/s when dehydrated (8.2 waters/lipid) and (1.9 +/- 0.1) x 10(-11) m(2)/s in excess water. The diffusion constant of water in dehydrated POPC was found to be (4.7 +/- 0.1) x 10(-10) m(2)/s. The radius of curvature is 21 +/- 2 microm for the dehydrated sample and 4.5 +/- 0.5 microm for POPC sample containing excess water. The activation energies of diffusion are 40.6 +/- 0.4 kJ/mole for dehydrated POPC, 30.7 +/- 0.9 kJ/mole for POPC with excess water, and 28.6 +/- 1.5 kJ/mole for water in dehydrated POPC. The diffusion constants and activation energies for a sample of POPC/ibuprofen/water (1:0.56:15) were also measured. The ibuprofen, which locates in the lipid-water interface, diffuses faster than POPC but has a slightly higher activation energy of lateral diffusion. Within certain restrictions, PFG-MAS NMR provides a useful method for characterizing membrane organization and mobility.     

The association of yeast phosphoglycerate kinase (EC 2.7.2.3).

1. A mol.wt. of 40030 +/- 830 has been estimated for phosphoglycerate kinase in concentrations less than 0.1 g/100 cm3 comparing favourably with expected values from X-ray diffraction measurements by 10% lower than the previously reported molecular weights made at higher concentrations. 2. The so20w, was estimated to be 3.12(+/-0.02)x10(-13)s and the coefficient had a low concentration dependency giving a g value (concentration-dependency) of 2.3 +/- 1.6cm3 .g-1. This agrees with previous qualitative observations. 3. By using fluctuation-intensity spectroscopy, the D20,w was estimated to be 7.4(+/-0.2)x10(-11)m2.s-1, and this was indistinguishable from the D20,w calculated from ultracentrifuge results. The water of hydration was estimated to be 0.46 g/g of protein. 4. It is inferred from the estimates that phosphoglycerate kinase associates with an interaction coefficient at 20 degrees C for monomer/dimer of between 10 and 12 cm3.g-1. 5. The ratio of molecular asymmetry (a/b) was estimated to be 2.5+/-0.2 from the values of D20,w and water of hydration. This compares favourably with the ratio from the overall dimensions estimated from X-ray diffraction measurements.     

Permeability of human granulocytes to water: rectification of osmotic flow

The permeability of human granulocytes to water was studied for samples of that population with an electronic particle counter. Changes in volume were monitored as these samples were introduced suddenly into hypo- and hypertonic osmotica. Temperature and concentration sensitivity analyses of the permeability coefficients were carried out. An apparent rectification of water flow seems to occur with the water permeability being three times higher for endosmotic flow than for exosmotic flow. The estimated water permeability at the reference temperature 20 degrees C was for exosmotic flow 2.04 +/- 0.02 x 10(-12) kgmol N-1 s-1 with an apparent transfer energy of 4.76 +/- 0.09 x 10(7) J kgmol-1, and for endosmotic flow 6.05 +/- 0.07 x 10(-12) kgmol N-1 s-1 with an apparent transfer energy of 4.9 +/- 0.1 x 10(7) J kgmol-1.     

Characterization of leukotriene B4 synthesis in canine polymorphonuclear leukocytes.

The synthesis and release of leukotriene B4 (LTB4) from canine polymorphonuclear leukocytes (PMNs) was characterized in terms of incubation time, temperature and effects of calcium ionophore A23187 concentrations. Maximal LTB4 concentrations were determined when canine PMNs were incubated with 10 microM A23187. Increasing LTB4 concentrations were determined through 10 min incubation. The maximal LTB4 concentrations (310 +/- 30 pg LTB4/2.5 x 10(5) cells) determined at 10 min did not change through a 55 min incubation period. Greater LTB4 concentrations were synthesized by canine PMNs at 37 degrees C (268 +/- 12 pg LTB4/2.5 x 10(5) cells) than at 25 degrees C (206 +/- 11 pg LTB4/2.5 x 10(5) cells) or 5 degrees C (59 +/- 3 pg LTB4/2.5 x 10(5) cells). The synthesis of LTB4 in canine PMNs was inhibited by incubation of the cells with either of two known lipoxygenase inhibitors, BWA4C or BW755C. BWA4C inhibited LTB4 synthesis with an approximate IC50 = 0.1 microM, whereas BW755C inhibited LTB4 synthesis with an approximate IC50 = 10 microM. These results indicate canine PMNs have the capability to synthesize large quantities of LTB4 when stimulated with calcium ionophore A23187. Furthermore, the 5-lipoxygenase inhibitors BWA4C, an acetohydroxyamic acid, and BW755C, a phenyl pyrazoline, can readily inhibit LTB4 synthesis in canine PMNs.     

The reduction of ferrate(VI) to ferrate(V) by ascorbate

The reduction of ferrate(VI) by ascorbate has been studied under anaerobic conditions in the pH range between 6.8 and 11.5 at 24 degrees C. A mechanism is proposed that is consistent with the observed rate constants k11 (HFeO4- + AH-) = (5.6 +/- 0.6) x 10(6) M-1 s-1, k12(FeO4(2-) + AH-) = (1.3 +/- 0.1) x 10(6) M-1 s-1 and the pK(HFeO4- in equilibrium with H(+) + FeO4(2-) = 7.9. Stoichiometric studies show that at high ratios of [AH-]/[FeO4(2-)], one ferrate(VI) oxidizes three molecules of ascorbate to the corresponding ascorbyl (A-) radicals.     

Optical measurement of osmotic water transport in cultured cells. Role of glucose transporters

Methodology was developed to measure osmotic water permeability in monolayer cultured cells and applied to examine the proposed role of glucose transporters in the water pathway (1989. Proc. Natl. Acad. Sci. USA. 86:8397-8401). J774 macrophages were grown on glass coverslips and mounted in a channel-type perfusion chamber for rapid fluid exchange without cell detachment. Relative cell volume was measured by 45 degrees light scattering using an inverted microscope; measurement accuracy was validated by confocal imaging microscopy. The time required for greater than 90% fluid exchange was less than 1 s. In response to a decrease in perfusate osmolality from 300 to 210 mosM, cells swelled without lag at an initial rate of 4.5%/s, corresponding to a water permeability coefficient of (6.3 +/- 0.4) x 10(-3) cm/s (SE, n = 20, 23 degrees C), assuming a cell surface-to-volume ratio of 4,400 cm-1. The initial rate of cell swelling was proportional to osmotic gradient size, independent of perfusate viscosity, and increased by amphotericin B (25 micrograms/ml), and had an activation energy of 10.0 +/- 1 kcal/mol (12-39 degrees C). The compounds phloretin (20 microM) and cytochalasin B (2.5 micrograms/ml) inhibited glucose transport by greater than 85% but did not influence Pf in paired experiments in which Pf was measured before and after inhibitor addition. The mercurials HgCl2 (0.1 mM) and p-chloromercuribenzoate (1 mM) did not inhibit Pf. A stopped-flow light scattering technique was used to measure Pf independently in J774 macrophages grown in suspension culture. Pf in suspended cells was (4.4 +/- 0.3) x 10(-3) cm/s (assuming a surface-to-volume ratio of 8,800 cm-1), increased more than threefold by amphotericin B, and not inhibited by phloretin and cytochalasin B under conditions of strong inhibition of glucose transport. The glucose reflection coefficient was 0.98 +/- 0.03 as measured by induced osmosis, assuming a unity reflection coefficient for sucrose. These results establish a quantitative method for measurement of osmotic water transport in adherent cultured cells and provide evidence that glucose transporters are not involved in the water transporting pathway.     

Production of Lactase by Candida pseudotropicalis Grown in Whey

Lactase (beta-d-galactosidase) was produced by Candida pseudotropicalis grown in deproteinized whey. Maximum enzyme production in 2% whey was obtained by supplementation with 0.15% yeast extract, 0.1% (NH(4))(2)SO(4), and 0.05% KH(2)PO(4) (wt/vol). Highest enzyme values (4.35 U/mg of cells and 68 U/ml) were obtained with 10 to 12% whey, while enzyme yield was maximal in 2% whey (0.87 U/mg of whey). Optimal initial pH for cultivation was 3.5. The best conditions for extraction included 2% (wt/vol) chloroform, 10 h of treatment, pH 6.6 and higher, and 30 to 37 degrees C. Optimum pH and temperature for enzyme activity were 6.2 and 47 degrees C. The enzyme had a K(m) for O-nitrophenyl-beta-d-galactopyranoside of 3.06 x 10 M and the initial V(max) was estimated as 6.63 x 10 M per min. It hydrolized 50 and 100% of the lactose in whey and milk within 4 and 5 h, respectively, at 37 degrees C. The lyophilized enzyme retained 95% of activity for 3 months when stored at -20 degrees C.     

Physiological characterization of Dunaliella sp. (Chlorophyta, Volvocales) from Yucatan, Mexico

Physiological responses of Dunaliella salina and Dunaliella viridis, isolated from solar saltworks on the Yucatan Peninsula, were studied. Optimal growth temperature for D. salina was 22 degrees C (3.06 x 10(6) cells mL(-1)) and 26 degrees C for D. viridis (4.04 x 10(6)cells mL(-1)). Total carotenoid content in D. salina increased with temperature to a maximum of 35.14 pg cell(-1) at 38 degrees C. Dunaliella salina alpha-carotene and beta-carotene content was 0.083+/-0.003 and 0.598+/-0.020 mg 100g dry wt(-1) respectively, whereas lower values were found in D. viridis cultured under same experimental conditions (0.018+/-0.002 and 0.136+/-0.012 mg 100g dry wt(-1) respectively). The highest specific growth rate in D. salina was obtained at 10% NaCl (0.28 d(-1)), while its cell volume increased from 524 to 2066.93 microm(3) when cultured from 10% to 35% NaCl. Maximum photosynthetic rates were attained when increasing from optimal growing temperature to 30 degrees C for D. viridis (108 n mol O(2)microg chl alpha h(-1)) and D. salina (139 n mol O(2)microg chl alpha h(-1)). Photosynthetic responses to temperature variations indicated physiological adjustments in both species, with higher acclimation in D. salina. Evaluation of physiological attributes of these species will be used for to carry out mass cultivation.     

Mechanism of electroporative dye uptake by mouse B cells.

The color change of electroporated intact immunoglobulin G receptor (Fc gammaR-) mouse B cells (line IIA1.6) after direct electroporative transfer of the dye SERVA blue G (Mr 854) into the cell interior is shown to be dominantly due to diffusion of the dye after the electric field pulse. Hence the dye transport is described by Fick's first law, where, as a novelty, time-integrated flow coefficients are introduced. The chemical-kinetic analysis uses three different pore states (P) in the reaction cascade (C <==> P1 <==> P2 <==> P3), to model the sigmoid kinetics of pore formation as well as the biphasic pore resealing. The rate coefficient for pore formation k(p) is dependent on the external electric field strength E and pulse duration tE. At E = 2.1 kV cm(-1) and tE = 200 micros, k(p) = (2.4 +/- 0.2) x 10(3) s(-1) at T = 293 K; the respective (field-dependent) flow coefficient and permeability coefficient are k(f)0 = (1.0 +/- 0.1) x 10(-2) s(-1) and P0 = 2 cm s(-1), respectively. The maximum value of the fractional surface area of the dye-conductive pores is 0.035 +/- 0.003%, and the maximum pore number is Np = (1.5 +/- 0.1) x 10(5) per average cell. The diffusion coefficient for SERVA blue G, D = 10(-6) cm2 s(-1), is slightly smaller than that of free dye diffusion, indicating transient interaction of the dye with the pore lipids during translocation. The mean radii of the three pore states are r(P1) = 0.7 +/- 0.1 nm, r(P2) = 1.0 +/- 0.1 nm, and r(P3) = 1.2 +/- 0.1 nm, respectively. The resealing rate coefficients are k(-2) = (4.0 +/- 0.5) x 10(-2) s(-1) and k(-3) = (4.5 +/- 0.5) x 10)(-3) s(-1), independent of E. At zero field, the equilibrium constant of the pore states (P) relative to closed membrane states (C) is K(p)0 = [(P)]/[C] = 0.02 +/- 0.002, indicating 2.0 +/- 0.2% water associated with the lipid membrane. Finally, the results of SERVA blue G cell coloring and the new analytical framework may also serve as a guideline for the optimization of the electroporative delivery of drugs that are similar in structure to SERVA blue G, for instance, bleomycin, which has been used successfully in the new discipline of electrochemotherapy.     

MR imaging measurement of compartmental water diffusion in perfused heart slices

Myocardial tissue slices were isolated from the left ventricular free wall (7 slices) and left ventricular papillary muscle (3 slices) of New Zealand White male rabbits (n = 4) and were subsequently superfused with a modified St. Thomas' Hospital cardioplegic solution at 19 degrees C. The diffusion-weighted images were obtained with a 600-MHz nuclear magnetic resonance spectrometer using diffusion gradient b-values that ranged from 166 to 6,408 s/mm(2); the apparent diffusion coefficient of water in the tissues were subsequently calculated. All of the tissue samples that were studied exhibited nonmonoexponential diffusion. Data from seven slices were mathematically fitted by a biexponential expression with a fast diffusion component of 0.72 +/- 0.07 x 10(-3) mm(2)/s, and a slow diffusion component of 0.060 +/- 0.033 x 10(-3) mm(2)/s. The fast component dominated the calculated apparent diffusion coefficient of the tissue, composed of 82 +/- 3% of the overall diffusion-dependent signal decay. Thus myocardial tissue exhibits characteristics consistent with multiple compartments of diffusion. This work has important implications for myocardial diffusion tensor imaging, as well as the changes in diffusion that have been reported following myocardial ischemia.     

Thermodynamic and kinetic characterization of Co-C bond homolysis catalyzed by coenzyme B(12)-dependent methylmalonyl-CoA mutase

Methylmalonyl-CoA mutase is a member of the family of coenzyme B(12)-dependent isomerases and catalyzes the 1,2-rearrangement of methylmalonyl-CoA to succinyl-CoA. A common first step in the reactions catalyzed by coenzyme B(12)-dependent enzymes is cleavage of the cobalt-carbon bond of the cofactor, leading to radical-based rearrangement reactions. Comparison of the homolysis rate for the free and enzyme-bound cofactors reveals an enormous rate enhancement which is on the order of a trillion-fold. To address how this large rate acceleration is achieved, we have examined the kinetic and thermodynamic parameters associated with the homolysis reaction catalyzed by methylmalonyl-CoA mutase. Both the rate and the amount of cob(II)alamin formation have been analyzed as a function of temperature with the protiated substrate. These studies yield the following activation parameters for the homolytic reaction at 37 degrees C: DeltaH(f)() = 18.8 +/- 0.8 kcal/mol, DeltaS(f)() = 18.2 +/- 0.8 cal/(mol.K), and DeltaG(f)() = 13.1 +/- 0.6 kcal/mol. Our results reveal that the enzyme lowers the transition state barrier by 17 kcal/mol, corresponding to a rate acceleration of 0.9 x 10(12)-fold. Both entropic and enthalpic factors contribute to the observed rate acceleration, with the latter predominating. The substrate binding step is exothermic, with a DeltaG of -5.2 kcal/mol at 37 degrees C, and is favored by both entropic and enthalpic factors. We have employed the available kinetic and spectroscopic data to construct a qualitative free energy profile for the methylmalonyl-CoA mutase-catalyzed reaction.     

The [Ru(Hedta)NO](0.1-) system: structure, chemical reactivity and biological assays

The [Ru(II)(Hedta)NO(+)] complex is a diamagnetic species crystallizing in a distorted octahedral geometry, with the Ru-N(O) length 1.756(4) A and the RuNO angle 172.3(4) degrees . The complex contains one protonated carboxylate (pK(a)=2.7+/-0.1). The [Ru(II)(Hedta)NO(+)] complex undergoes a nitrosyl-centered one-electron reduction (chemical or electrochemical), with E(NO+/NO)=-0.31 V vs SCE (I=0.2 M, pH 1), yielding [Ru(II)(Hedta)NO](-), which aquates slowly: k(-NO)=2.1+/-0.4x10(-3) s(-1) (pH 1.0, I=0.2 M, CF(3)COOH/NaCF(3)COO, 25 degrees C). At pHs>12, the predominant species, [Ru(II)(edta)NO](-), reacts according to [Ru(II)(edta)NO](-)+2OH(-)-->[Ru(II)(edta)NO(2)](3-), with K(eq)=1.0+/-0.4 x 10(3) M(-2) (I=1.0 M, NaCl; T=25.0+/-0.1 degrees C). The rate-law is first order in each of the reactants for most reaction conditions, with k(OH(-))=4.35+/-0.02 M(-1)s(-1) (25.0 degrees C), assignable mechanistically to the elementary step comprising the attack of one OH(-) on [Ru(II)(edta)NO](-), with subsequent fast deprotonation of the [Ru(II)(edta)NO(2)H](2-) intermediate. The activation parameters were DeltaH(#)=60+/-1 kJ/mol, DeltaS(#)=-31+/-3 J/Kmol, consistent with a nucleophilic addition process between likely charged ions. In the toxicity up-and-down tests performed with Swiss mice, no death was observed in all the doses administered (3-9.08 x 10(-5) mol/kg). The biodistribution tests performed with Wistar male rats showed metal in the liver, kidney, urine and plasma. Eight hours after the injection no metal was detected in the samples. The vasodilator effect of [Ru(II)(edta)NO](-) was studied in aortic rings without endothelium, and was compared with sodium nitroprusside (SNP). The times of maximal effects of [Ru(II)(edta)NO](-) and SNP were 2 h and 12 min, respectively, suggesting that [Ru(II)(edta)NO](-) releases NO slowly to the medium in comparison with SNP.     

cAMP regulated membrane diffusion of a green fluorescent protein-aquaporin 2 chimera

To study the membrane mobility of aquaporin water channels, clones of stably transfected LLC-PK1 cells were isolated with plasma membrane expression of GFP-AQP1 and GFP-AQP2, in which the green fluorescent protein (GFP) was fused upstream and in-frame to each aquaporin (AQP). The GFP fusion did not affect AQP tetrameric association or water transport function. GFP-AQP lateral mobility was measured by irreversibly bleaching a spot (diameter 0.8 microm) on the membrane with an Argon laser beam (488 nm) and following the fluorescence recovery into the bleached area resulting from GFP translational diffusion. In cells expressing GFP-AQP1, fluorescence recovered to >96% of its initial level with t(1/2) of 38 +/- 2 s (23 degrees C) and 21 +/- 1 s (37 degrees C), giving diffusion coefficients (D) of 5.3 and 9.3 x 10(-11) cm(2)/s. GFP-AQP1 diffusion was abolished by paraformaldehyde fixation, slowed >50-fold by the cholesterol-binding agent filipin, but not affected by cAMP agonists. In cells expressing GFP-AQP2, fluorescence recovered to >98% with D of 5.7 and 9.0 x 10(-11) cm(2)/s at 23 degrees C and 37 degrees C. In contrast to results for GFP-AQP1, the cAMP agonist forskolin slowed GFP-AQP2 mobility by up to tenfold. The cAMP slowing was blocked by actin filament disruption with cytochalasin D, by K(+)-depletion in combination with hypotonic shock, and by mutation of the protein kinase A phosphorylation consensus site (S256A) at the AQP2 C-terminus. These results indicate unregulated diffusion of AQP1 in membranes, but regulated AQP2 diffusion that was dependent on phosphorylation at serine 256, and an intact actin cytoskeleton and clathrin coated pit. The cAMP-induced immobilization of phosphorylated AQP2 provides evidence for AQP2-protein interactions that may be important for retention of AQP2 in specialized membrane domains for efficient membrane recycling.     

Ontogeny of rabbit proximal tubule urea permeability

Urea transport in the proximal tubule is passive and is dependent on the epithelial permeability. The present study examined the maturation of urea permeability (P(urea)) in in vitro perfused proximal convoluted tubules (PCT) and basolateral membrane vesicles (BLMV) from rabbit renal cortex. Urea transport was lower in neonatal than adult PCT at both 37 and 25 degrees C. The PCT P(urea) was also lower in the neonates than the adults (37 degrees C: 45.4 +/- 10.8 vs. 88.5 +/- 15.2 x 10(-6) cm/s, P < 0.05; 25 degrees C: 28.5 +/- 6.9 vs. 55.3 +/- 10.4 x 10(-6) cm/s; P < 0.05). The activation energy for PCT P(urea) was not different between the neonatal and adult groups. BLMV P(urea) was determined by measuring vesicle shrinkage, due to efflux of urea, using a stop-flow instrument. Neonatal BLMV P(urea) was not different from adult BLMV P(urea) at 37 degrees C [1.14 +/- 0.05 x 10(-6) vs. 1.25 +/- 0.05 x 10(-6) cm/s; P = not significant (NS)] or 25 degrees C (0.94 +/- 0.06 vs. 1.05 +/- 0.10 x 10(-6) cm/s; P = NS). There was no effect of 250 microM phloretin, an inhibitor of the urea transporter, on P(urea) in either adult or neonatal BLMV. The activation energy for urea diffusion was also identical in the neonatal and adult BLMV. These findings in the BLMV are in contrast to the brush-border membrane vesicles (BBMV) where we have previously demonstrated that urea transport is lower in the neonate than the adult. Urea transport is lower in the neonatal proximal tubule than the adult. This is due to a lower rate of apical membrane urea transport, whereas basolateral urea transport is the same in neonates and adults. The lower P(urea) in neonatal proximal tubules may play a role in overall urea excretion and in developing and maintaining a high medullary urea concentration and thus in the ability to concentrate the urine during renal maturation.     

Intercalation of proflavine and a platinum derivative of proflavine into double-helical Poly(A)

The equilibria and kinetics of the interactions of proflavine (PR) and its platinum-containing derivative [PtCl(tmen)(2)HNC(13)H(7)(NHCH(2)CH(2))(2)](+) (PRPt) with double-stranded poly(A) have been investigated by spectrophotometry and Joule temperature-jump relaxation at ionic strength 0.1 M, 25 degrees C, and pH 5.2. Spectrophotometric measurements indicate that base-dye interactions are prevailing. T-jump experiments with polarized light showed that effects due to field-induced alignment could be neglected. Both of the investigated systems display two relaxation effects. The kinetic features of the reaction are discussed in terms of a two-step series mechanism in which a precursor complex DS(I) is formed in the fast step, which is then converted to a final complex in the slow step. The rate constants of the fast step are k(1) = (2.5 +/- 0.4) x 10(6) M(-1) s(-1), k(-1) = (2.4 +/- 0.1) x 10(3) s(-1) for poly(A)-PR and k(1) = (2.3 +/- 0.1) x 10(6) M(-1) s(-1), k(-1) = (1.6 +/- 0.2) x 10(3) s(-1) for poly(A)-PRPt. The rate constants for the slow step are k(2) = (4.5 +/- 0.5) x 10(2) s(-1), k(-2) = (1.7 +/- 0.1) x 10(2) s(-1) for poly(A)-PR and k(2) = 9.7 +/- 1.2 s(-1), k(-2) = 10.6 +/- 0.2 s(-1) for poly(A)-PRPt. Spectrophotometric measurements yield for the equilibrium constants and site size the values K = (4.5 +/- 0.1) x 10(3) M(-1), n = 1.3 +/- 0.5 for poly(A)-PR and K = (2.9 +/- 0.1) x 10(3) M(-1), n = 2.3 +/- 0.6 for poly(A)-PRPt. The values of k(1) are similar and lower than expected for diffusion-limited reactions. The values of k(-1) are similar as well. It is suggested that the formation of DS(I) involves only the proflavine residues in both systems. In contrast, the values of k(2) and k(-2) in poly(A)-PRPt are much lower than in poly(A)-PR. The results suggest that in the complex DS(II) of poly(A)-PRPt both proflavine and platinum residues are intercalated. In addition, a very slow process was detected and ascribed to the covalent binding of Pt(II) to the adenine.     

Optimal conditions for the production of sulfated polysaccharide by marine microalga Gyrodinium impudicum strain KG03

A marine microalga Gyrodinium impudicum strain KG03 produced sulfated exopolysaccharide designated as p-KG03, which showed a strong antiviral activity against encephalomyocarditis virus (EMCV). To optimize culture conditions for the production of p-KG03, mineral salts, vitamins, plant growth hormones, temperature, pH and light conditions were examined. From this study, M-KG03 medium for the maximum production of p-KG03 was suggested as follows; NH(4)Cl 75 microM, NaH(3)PO(4) 200 microM, NaHCO(3) 50 microM, Na(2)SO(4) 10 microM, FeCl(2) x 6H(2)O 10 microM, MnCl(2) x 4H(2)O 0.1 microM, vitamin B(12) 0.75 microg, naphthalene acetic acid (NAA) 7.5 microg and myo-inositol 200 mg per liter of aged sea water. The optimal temperature and pH were 22.5 degrees C and 8.0, respectively. The optimal light conditions of intensity and period were 150 microE m(-2) s(-1) and 16:8 h light:dark cycle. Finally, the cell growth and p-KG03 production were measured in one liter of M-KG03 medium with 1% CO(2) and 50 ml min(-1) of airflow using two liters airlift balloon type photobioreactor (ABTPR). At these optimal conditions, p-KG03 production and cell growth were 134.6+/-5.9 mg l(-1) and 123,076+/-1,597 cells ml(-1), respectively, representing a 7.7 and 5.1 times compared with f/2 medium with Erlenmeyer flask culture (p-KG03 production 17.5+/-1.3 mg l(-1) and cell growth 24,311+/-1,291 cells ml(-1)).     

Diffusion, patching, and capping of stearoylated dextrans on 3T3 cell plasma membranes

Fluorescence-labeled trinitrophenylated stearoylated dextrans have been used as controllable analogues of cell membrane proteins on model membranes and on a variety of natural cell membranes. This paper reports their behavior on 3T3 mouse fibroblast plasma membranes. Spatial distribution on the membrane was studied by fluorescence microscopy, and molecular mobility was measured by fluorescence photobleaching recovery. At concentrations from 10(2) to 3 X 10(3) molecules/micron2 essentially homogeneous fluorescence was observed after treatment with these stearoyldextrans in culture. Diffusion coefficients and fractional recovery of fluorescence after photobleaching were cvoncentration independent. For 3 X 10(3) molecules/micron2 we found at 23 degrees C D = (3.0 +/- 1.8) X 10(-10) cm2/s with 65 +/- 17% recovery and at 37 degrees C D = (7.0 +/- 5.0) X 10(-10) cm2/s without a change of the fractional recovery. Cross-linking with antibodies stopped diffusion on a macroscopic scale and sometimes induced patching, mottling (defined as the development of gaps in the fluorescence layer), and capping (defined as the confinement of the fluorescence to less than 50% of the cell). Capping required approximately 3 h at 37 degrees C and was inhibited by metabolic poisons and cytochalasin B. These drugs did not affect stearoyldextran diffusion or fractional recovery. Colchicine, which did not dramatically affect capping, slowed diffusion two- to threefold but did not affect fractional recovery. The antibody inhibition of the diffusion of stearoyldextrans precedent to capping did not affect the diffusion of a lipid probe or fluorescein isothiocyanate labeled membrane proteins. When the trinitrophenylated stearoyldextran was cleared from most of the surface by capping and the surface subsequently relabeled with stearoyldextran, the diffusion coefficient and fractional recovery of the second label were identical with those of the first label prior to capping. Thus, capping does not clear an immobilizing factor from the membrane.     

Membrane formation and drug loading effects in high amylose starch tablets studied by NMR imaging

Cross-linked high amylose starch is used as an excipient in the preparation of pharmaceutical tablets for the sustained release of drugs. NMR imaging with contrast enhanced by proton density and by self-diffusion coefficient was used to follow the water uptake and swelling, two critical parameters controlling the drug release of the cross-linked starch tablets containing 10 wt % of ciprofloxacin and of acetaminophen, respectively. The drug-loaded tablets were studied in a H2O/D2O mixture at 37 degrees C in comparison to the tablets without any drug loading. The diffusion of water in the tablets all showed a Fickian behavior, but the kinetics of water uptake was faster in the case of the drug-loaded tablets. The formation of a membrane at the water/tablet interface was observed.     

Diffusion coefficients in the lateral intercellular spaces of Madin-Darby canine kidney cell epithelium determined with caged compounds.

The diffusion coefficients of two caged fluorescent dyes were measured in free solution and in the lateral intercellular spaces (LIS) of cultured Madin-Darby canine kidney (MDCK) cells after photoactivation by illumination with a continuous or pulsed UV laser. Both quantitative video imaging and a new photometric method were utilized to determine the rates of diffusion of the caged fluorescent dyes: 8-((4,5-dimethoxy-2-nitrobenzyl)oxy)pyrene-1,3,6-trisulfonic acid (DMNB-HPTS) and (4,5-dimethoxy-2-nitrobenzyl) fluorescein dextran (10,000 MW) (DMNB-caged fluorescein dextran). The diffusion coefficients at 37 degrees C in free solution were 3.3 x 10(-6) cm2/s (HPTS) and 0.98 x 10(-6) cm2/s (10,000 MW dextran). Diffusion of HPTS within nominally linear stretches of the LIS of MDCK cells grown on glass coverslips was indistinguishable from that in free solution, whereas dextran showed a 1.6 +/- 0.5-fold reduction in diffusivity. Measurements of HPTS diffusion within the LIS of multicellular regions also exhibited a diffusivity comparable to the free solution value. The restriction to diffusion of the dextran within the LIS may be due to molecular hindrance.     

Preservation of tomcat (Felis catus) semen in variable temperatures

The aim of our study was to estimate the viability of cat epididymal sperm in short time storage at +4 degrees C and in long term storage at -196 degrees C and to assess the percentage of live sperm in fresh semen using eosin/nigrosin staining compared to the flow cytometry method. The testes with epididymides were obtained after routine castration procedure. The sperm for further research were collected after flushing the epididymides using extender consist of: Tris 2.4 g, citric acid 1.4 g, glucose 0.8 g, 0.06% (w/v) Na-benzylpenicillin, 0.1% (w/v) streptomycin sulphate and distilled water. Half of each sample was equilibrated with the dilution and loaded in 0.25 ml plastic straws. The straws were placed on a rack in liquid nitrogen vapour at -120 degrees C for 10 min, plunged in liquid nitrogen for 10 min, replaced to marked goblets and loaded into canes for long term storage in liquid nitrogen at -196 degrees C. Sixty percent of motile spermatozoa was accomplished after thawing. However, the percentage of the sperm with intact acrosomes was decreased and the share of cells with midpiece and tail defects was increased. The storage of sperm flushed from epididymides at +4 degrees C for a short time and the usage of sperm during 2-3 days after collection seems to be better than cryopreservation. In our study, normospermia was present in 72.7 +/- 8.8% of fresh semen. The most common defect was the presence of distal droplets, imperfect heads or abnormal acrosomal outline. The motility of fresh sperm flushed from epididymides achieved 77.9 +/- 6.8%. The viability of sperm amounting to 52.5 +/- 13.8% was achieved on third day of conservation in the liquid extender. The percentage of viable sperm in fresh epididymal spermatozoa was 84.9 +/- 7.8%. Compared to these results, the percentage of live cells using SYBR-14/propidium iodide staining was insignificantly lower (82.2 +/- 8%). The live, non-apoptotic cells were 79.0 +/- 7.8%. The share of live, early-apoptotic spermatozoa and late-apoptotic spermatozoa was, respectively, 2 +/- 1.4% and 1.5 +/- 0.9%. The viability of sperm estimated by eosin/nigrosin staining was confirmed by the flow cytometry method. There was no statistical differences between the staining. The usage of apoptosis detection kit revealed, that the percentage of early-apoptotic and late-apoptotic cells was insignificant.