Factors affecting DNA binding and stability of association to cationic liposomes

Lipoplexes are complexes formed between cationic liposomes (L(+)) and polyanionic nucleic acids (P(-)). They are commonly used in vitro and in vivo as a nucleic acid delivery system. Our study aims are to investigate how DOTAP-based cationic liposomes, which vary in their helper lipid (cholesterol or DOPE) and in media of different ionic strengths affect the degree, mode of association and degree of condensation of pDNA. This was determined by ultracentrifugation and gel electrophoresis, methods based on different physical principles. In addition, the degree of pDNA condensation was also determined using the ethidium bromide (EtBr) intercalation assay. The results suggest that for cationic lipid compositions (DOTAP/DOPE and DOTAP/cholesterol), 1.5 M NaCl, but not 0.15 M NaCl, both prevent lipoplex formation and/or induce partial dissociation between lipid and DNA of preformed lipoplexes. The higher the salt concentration the greater is the similarity of DNA condensation (monitored by EtBr intercalation) between lipoplex DNA and free DNA. As determined by ultracentrifugation and agarose gel electrophoresis, 30-90% of the DNA is uncondensed. SDS below its critical micellar concentration (CMC) induced "de-condensation" of DNA without its physical release (assessed by ultracentrifugation) for both DOTAP/DOPE and DOTAP/cholesterol lipoplexes. As was assessed by agarose gel electrophoresis SDS induced release of 50-60% of DNA from the DOTAP/cholesterol lipoplex but not from the DOTAP/DOPE lipoplex. This study shows that there are conditions under which DNA is still physically associated with the cationic lipids but undergoes unwinding to become less condensed. We also proved that the helper lipid affects level and strength of the L(+) and DNA(-) electrostatic association; these interactions are weaker for DOTAP/cholesterol than for DOTAP/DOPE, despite the fact that the positive charge and surface pH of DOTAP/cholesterol and DOTAP/DOPE are similar.     

Factors affecting the encapsulation of drugs within liposomes.

The reported efficiencies of drug encapsulation into liposomes range from less than 0.1% to more than 10% per micromole phospholipid, depending on the nature of the drug and of the liposome employed. We have sought to investigate some of the factors which control the efficiency of drug encapsulation. We have found that most polar drugs are sequestered within the internal aqueous compartment of the liposomes, while nonpolar drugs can bind to the liposome membrane in addition to being sequestered, thus accounting for their higher efficiencies of encapsulation. The encapsulation of nonpolar drugs, but not of polar drugs, is very sensitive to the physical characteristics of the liposome membrane; in particular, a fluid membrane favors the efficient encapsulation of nonpolar compounds. The drug cytosine arabinoside is anomalous in that this highly polar compound seems to interact with the liposome membrane at physiological conditions of pH and ionic strength, thus allowing it to be encapsulated with high efficiency.     

Factors affecting the character stability of classifications

The effect of six resemblance coefficients (taxonomic distance, Manhattan distance, correlations, cosines, and two new general dissimilarity coefficients) on the character stability of classifications based on six data sets was evaluated. The six data sets represent a variety of organisms, and of ratios of number of characters to number of OTUs, and were randomly bipartitioned 100 times. The results of matrix correlations, cophenetic correlations and two consensus measures indicate that no one resemblance coefficient is uniformly better than all others when evaluated in terms of the stability of a classification, although taxonomic distance and Manhattan distance produce relatively more stable classifications than the other resemblance coefficients. An index of dimensionality, the stemminess and cophenetic correlations of classifications were calculated for the six data sets and also for 20 data sets analyzed in an earlier study. Regression analysis of stability on the ratio of number of characters to the number of OTUs, dimensionality, stemminess, and cophenetic correlations explained more than 70% of the variance in stability. Of the four factors, the ratio was by far the most important. Stemminess and dimensionality contributed little when considered singly, and did not add appreciably to the variance explained by ratio and cophenetic correlations.Dedicated to the memory of Prof.J. S. L. Gilmour. His insightful wrightings on naturalness in classifications paved the way for the development of numerical phenetics.     

Factors affecting the stability of cryogenically preserved granulocytes

Human granulocytes free of other cell types were obtained by counterflow centrifugation, cryogenically preserved, and studied for stability and function after thawing.Isolation of granulocytes by counterflow centrifugation was optimal at reduced temperatures (4–10 °C) in phosphate-buffered saline (or Ca²⁺-free buffers) at pH 7.1. A stabilizing protein, or HES was required. Routinely, 1.2% human or bovine serum albumin was used. Hyperosmolar (310 m0sm) buffers and post isolation handling in ice water baths was optimal for cryogenic preservation. Addition of DMSO at 22 °C produced transient shrinkage initially which depended on the rate of addition, concentration, and temperature. Within 10–15 min granulocytes returned to volume, but continued to swell, equilibrating for 1 hr at 20% larger volume. Ethidium uptake gradually increased. After 24 hr, extreme swelling, lysis, and ethidium uptake was observed at the highest concentration (10%) of DMSO. DMSO-induced swelling was prevented with HES.Granulocytes (30 × 10⁶ ? 50 × 10⁶) were frozen in 2.0-ml volumes in plastic tubes. The combination of 5% DMSO, 6% HES, 4% albumin, 0.056 M glucose in NormosolR at pH 7.1 produced the best yields. Granulocytes were first cooled to 4 °C, then to ?80 °C (approx rate 4 °C per min) in a mechanical freezer and finally stored in liquid nitrogen. Storage varied from days to months. Granulocytes were thawed at 42 °C by manually twirling the freezing tubes and they were subsequently maintained in ice water. They were diluted 3:1 dropwise with a room temperature solution of 7% HES, 1.2% albumin, and 0.026 M glucose in Normosol. Particle ingestion tests were conducted by incubation at room temperature for forty minutes with yeast or zymosan opsonized with autologous serum. Particles ingested were counted by microfluorimetry after two washings at 150g.Granulocytes could not be cryogenically preserved in plasma or serum. Heating or prefreezing of serum was ineffective, but dialysis or addition of EDTA overcame the destructive effect of serum. Neither treatment was an improvement over the standard freeze procedure using buffered albumin and cryoprotective components. β-mercaptoethanol added to the freezing medium caused the production of a single homogeneous population of osmotically inert, nonviable, ethidium-reactive granulocytes. This suggests that osmoregulation by granulocyte membranes is a critical requirement for cryopreservation.Preservation efficiency is species dependent, increasing in the order of human, baboon, guinea pig, and dog. Dog granulocytes can be stored for at least 8 months in liquid nitrogen with small loss of cells and functionality.The present efficiency of preservation of human granulocytes for 3–4 weeks of liquid nitrogen storage is 90–100% morphological and 40% functional recovery. Attempts to increase stability of thawed granulocytes with other additions to our current procedure have so far proved fruitless. These have consisted of inosine, adenine, pyruvate, gluconate, vitamin C, β-mercaptoethanol, para-phenylmethyl-sulfonylfluoride, and mannitol.     

Factors affecting dry weight accumulation in developing barley endosperm

Some factors that may be concerned in determining final grain weight in barley ( Hordeum vulgare L. var. distichum ) have been investigated. Variation in endosperm fresh and dry weight, volume and starch content have been recorded at different stages of grain development between anthesis and harvest-ripeness for two barleys, cvs Kym and Golden Promise, differing in final grain weight. Results were recorded under both field and glasshouse conditions. The results suggest that the higher final dry weight of Kym, in comparison with Golden Promise, is a function of both rate and duration of grain filling. Only at later stages of endosperm development did the differences in volume become significant and the Kym endosperms continued to increased rapidly in volume for two to three days after endosperm volume had reached a maximum in Golden Promise. The rates of starch accumulation in both cultivars were very similar but starch deposition continued in Kym endosperms for four to five days after deposition in Golden Promise endosperms had slowed down.     

Factors affecting postural stability of healthy young adults

The objective of this paper was to examine the relationship between body balancing functions and body characteristics, motor abilities and reaction time. Subjects were 33 university students and 11 professional basketball players sorted into four groups of athletic and non-athletic women and men. Each group consisted of eleven subjects. The body height, weight was measured and the body mass index (BMI) calculated. A bioelectrical device computed the body fat (%). Static and dynamic motor tests, as well as static and dynamic balance tests were used. The reaction time (RT) to sound and light stimuli was measured. The regression analysis of the data revealed significant linear relationship between the amplitude of body sways (BS) and BMI in all groups. Also high correlation was found between back muscle strength and BS in all groups except the non-athletic women. Negative correlation was found between endurance capacity and BS in basketball players, i.e. at higher endurance capacity smaller amplitude BS occurred (r = -0.620, p < 0.04). The RT values showed significant correlations with BS only in the basketball players (r = 0.620, p < 0.04). It is concluded that increase in BMI, back muscle strength and endurance capacity is associated with better postural stability. Some motor abilities (hip flexibility, vertical jumping) show no significant correlations with body balancing, while other motor performances (static hanging) and RT values correlate well with BS only in the well-trained elite basketball players.     

Factors affecting the stability of methanol dehydrogenase from Paracoccus denitrificans

Methanol dehydrogenase from Paracoccus denitrificans was purified to homogeneity in two steps from the periplasmic fraction of methanol-grown cells. The enzyme was composed of subunits of M(r) 67,000 and 12,000, and non-covalently bound pyrroloquinoline quinone. It exhibited a pH optimum at pH values of 9.0 and above. It was not stable at pH greater than 9.0, but exhibited little loss of activity after prolonged incubation at pH values as low as 4.5. Methyl dehydrogenase was relatively stable to thermal denaturation. The thermal stability was enhanced by the presence of Ca2+ and diminished by the presence of EDTA. These data suggest a structural role for Ca2+ in this enzyme, similar to what has been observed with quinoprotein glucose and ethanol dehydrogenases.     

Factors affecting the thermodynamic stability of small asymmetric internal loops in RNA

Optical melting experiments were used to determine the thermodynamic parameters for oligoribonucleotides containing small asymmetric internal loops. The results show a broad range of thermodynamic stabilities, which depend on loop size, asymmetry, sequence, closing base pairs, and length of helix stems. Imino proton NMR experiments provide evidence for possible hydrogen bonding in GA and UU mismatches in some asymmetric loops. The stabilizing effects of GA, GG, and UU mismatches on the thermodynamic stability of internal loops vary depending on the size and asymmetry of the loop. The dependence of loop stability on Watson-Crick closing base pairs may be explained by an account of hydrogen bonds. Models are presented for approximating the free energy increments of 2 x 3 and 1 x 3 internal loops.     

Factors affecting the stability and accuracy of the bioassay for the sperm attractant sirenin

Optimal response of the sperm of Allomyces from the highly male strain M16 to the chemotactic agent, sirenin, was shown to occur when the sperm suspension contained 2 mm piparazine-N', N-bis[2-ethane sulfonic acid] buffer, 3 mm CaCl(2), and chelated trace elements. For the male strain M3, the CaCl(2) needed was 3.5 mm with the other two components the same as for M16. The inclusion in the sperm suspension of MgCl(2), KH(2)PO(4), or NH(4)Cl was without effect, except that under certain conditions phosphate was detrimental. The variability of 10 replicate assays was substantially reduced by using sperm in the bioassay at a concentration of 500,000 per ml rather than the former concentration of 100,000 per ml with a concomitant reduction in the concentration of sirenin above the membrane to which the sperm were attracted.     

Factors affecting the activity and stability of the palmitoyl-coenzyme A hydrolase of rat brain

Palmitoyl-CoA hydrolase (EC 3.1.2.2) catalyses the irreversible hydrolysis of long-chain acyl-CoA thioesters. This enzyme is found primarily in the postmicrosomal supernatant fraction prepared from homogenates of rat brain. Either of two forms of the hydrolase, a lower-molecular-weight species of approx. 70000 or a higher-molecular-weight species of approx. 130000 can be isolated by gel filtration. The higher-molecular-weight form is obtained from columns of Sephadex G-200 eluted with buffer containing 10mum-palmitoyl-CoA or 20% (v/v) glycerol, whereas the lower-molecular-weight form is obtained when the eluting buffer does not contain palmitoyl-CoA or glycerol. The two forms of the hydrolase have the same pH optimum of 7.5, are equally sensitive to the thiol-blocking reagents p-hydroxymercuribenzoate, HgCl(2), and 5,5'-dithiobis-(2-nitrobenzoic acid), and exhibit the same K(m) (1.8mum) with palmitoyl-CoA as substrate. The two forms differ in the availability or reactivity of certain external thiol groups, as determined by covalent chromatography with activated thiol Sepharose. Dilute solutions of the lower-molecular-weight form of the hydrolase rapidly lose activity (50% in 60min at 0 degrees C), but there is no change in the K(m) with palmitoyl-CoA as substrate during this progressive inactivation. Dilutions of the hydrolase in buffer containing 10mum-palmitoyl-CoA retain full activity. However, addition of palmitoyl-CoA to solutions of the lower-molecular-weight form will not restore previously lost hydrolase activity. The evidence supports the conclusion that the substrate palmitoyl-CoA promotes the formation of a relatively stable dimer from two unstable subunits. This process may not be reversible, since the removal of palmitoyl-CoA or glycerol from solutions of the higher-molecular-weight form does not result in the appearance of the lower-molecular-weight form of the hydrolase.     

Factors encoded by and affecting the holding stability of yeast plasmid pSR1

Summary A 2 m DNA-like plasmid, pSR1, isolated from a strain of Zygosaccharomyces rouxii has three coding frames, P, S and R. Insertional inactivation of R completely abolished the intramolecular recombination, and the defect was complemented by an intact R frame on a coexistent plasmid molecule. The P and S regions were also transactive and important, but not essential, for the stable maintenance of the plasmid molecules. Insertional disruption of the P frame suggested that it produces a protein factor. Similar insertional disruption of the S frame affected the plasmid stability in Z. rouxii and Saccharomyces cerevisiae hosts differently, depending on whether the inserted DNA fragment was a short 8 bp SalI linker or a long (2.2 kb) DNA fragment. Results strongly suggested that the S region encodes two factors, one RNA and the other a protein, and that the S protein is compatible with a sprecific hostfactor in Z. rouxii, but not in S. cerevisiae. In addition, a cis-acting locus, Z, was found at a site in the plasmid molecule where no distinct open reading frames were located. No long direct repeats or inverted repeats were observed in the Z region, such as are found in the REP3 locus of 2 m DNA.     

Yeast alpha-isopropylmalate isomerase. Factors affecting stability and enzyme activity.

Yeast alpha-isopropylmalate isomerase was found to be markedly stabilized by high concentrations of glycerol and (NH4)2SO4. Such conditions of high ionic strength inhibited the enzyme, stabilized the enzyme to heat, and affected kinetic parameters. The isomerase was found to exhibit ionic strength-dependent hysteresis when enzyme, totally but reversibly inhibited by storage under conditions of high ionic strength of (NH4)2SO4, was transferred to a lower concentration of (NH4)2SO4. Alpha-Isopropylmalate isomerase was found to be sensitive to KCN and certain other chelators. The inactivation by KCN was prevented by high concentrations of (NH4)2SO4. These observations implicated a metal involvement but the nature of the metal was not revealed. The metal involvement and some of the other properties of alpha-isopropylmalate isomerase reveal a similarity to aconitase. The similarities in properties between the isomerase and aconitase are summarized. Studies of yeast alpha-isopropylmalate isomerase indicated that it is a single polypeptide of about Mr = 90,000.     

Factors affecting production and stability of the AcAFP antifungal peptide secreted by Aspergillus clavatus

We have previously reported the identification of a small, basic and cysteine-rich antifungal peptide (AcAFP) secreted by Aspergillus clavatus and shown its ability to prevent growth of various human- and plant-pathogenic filamentous fungi. In this study, we sought to determine the physiological/microbiological requirements to enhance the AcAFP production and the conditions influencing its stability. The maximum of AcAFP production was obtained when A. clavatus was grown on 2% glycerol as sole carbon source and 100 mM NaCl. The AcAFP expression was shown to be influenced by pH, being suppressed under acidic (pH 5) and strongly induced under alkaline conditions. The activity of the purified AcAFP was not affected by temperature; it loosed approximately 20% of its activity after 3 h at 100°C and was efficient through a large pH range (pH 5-12) with an optimum at pH 8. AcAFP activity decreased at high ionic strength and in the presence of 10 mM of divalent cations (Mn²⁺, Fe²⁺ and Ca²⁺).     

Stability of dry liposomes in sugar glasses.

Sugars, particularly trehalose and sucrose, are used to stabilize liposomes during hydration (freeze-drying and air-drying). As a result, dry liposomes are trapped in a sugar glass, a supersaturated and thermodynamically unstable solid solution. We investigated the effects of the glassy state on liposome fusion and solute retention in the dry state. Solute leakage from dry liposomes was extremely slow at temperatures below the glass transition temperature (Tg); however, it increased exponentially as temperature increased to near or above the Tg, indicating that the glassy state had to be maintained for dry liposomes to retain trapped solutes. The leakage of solutes from dry liposomes followed the law of first-order kinetics and was correlated linearly with liposome fusion. The kinetics of solute leakage showed an excellent fit with the Arrhenius equation at temperatures both above and below the Tg, with a transitional break near the Tg. The activation energy of solute leakage was 1320 kJ/mol at temperatures above the Tg, but increased to 1991 kJ/mol at temperatures below the Tg. The stabilization effect of sugar glass on dry liposomes may be associated with the elevated energy barrier for liposome fusion and the physical separation of dry liposomes in the glassy state. The half-life of solute retention in dry liposomes may be prolonged by storing dry liposomes at temperatures below the Tg and by increasing the Tg of the dry liposome preparation.     

恶梦的影响因素研究

恶梦是与睡眠相关的常见心理现象.目前,恶梦的概念仍具有争议,但恶梦与心理健康的关系,已经引起了研究者广泛关注.恶梦的病因学研究表明,恶梦与遗传及生理因素有关;恶梦具有人格倾向性,应激与创伤因素是其产生的主要原因,与分裂症等精神疾病高度相关;恶梦与睡眠呼吸相关因素关系不是很明确;恶梦与多巴胺受体激动剂等药物显著相关,恶梦产生的其他因素研究尚待发现.然而这些因素与恶梦的因果关系并不确定,各因素之间的关系及作用机制有待进一步研究.未来的研究可以改进研究方法,扩大研究对象,进行纵向研究等.     

Controlled human RBC modifications affecting the binding of cationic liposomes

Cationic liposomes were prepared either by sonication or by detergent dialysis and used to deliver the antioxidative enzyme glutathione peroxidase into human erythrocytes in vitro. The enrichment ability of these two preparations was similar, amounting to about 30% of the control cells. The lysis of enzyme-treated erythrocytes induced by photoirradiation in the presence of PPIX was compared with that of cells incubated with empty liposomes. Erythrocytes enriched with GPX appear to be more resistant toward photohemolysis. Pre-treatment of cells with neuraminidase or proteinase K suggests that: a) sialic acid seems to be essential for the cell-liposome fusion process, no enrichment being found with the neuraminidase-treated cells; b) hydrolysis of the outer membrane proteins leads to an increased fragility with respect to controls even in GPX-enriched cells. These results were confirmed by extrinsic fluorescence polarization experiments, using isolated erythrocyte membranes and specific fluorescent probes.