Baroreflex modulation of muscle sympathetic nerve activity during posthandgrip muscle ischemia in humans.

To identify whether muscle metaboreceptor stimulation alters baroreflex control of muscle sympathetic nerve activity (MSNA), MSNA, beat-by-beat arterial blood pressure (Finapres), and electrocardiogram were recorded in 11 healthy subjects in the supine position. Subjects performed 2 min of isometric handgrip exercise at 40% of maximal voluntary contraction followed by 2.5 min of posthandgrip muscle ischemia. During muscle ischemia, blood pressure was lowered and then raised by intravenous bolus infusions of sodium nitroprusside and phenylephrine HCl, respectively. The slope of the relationship between MSNA and diastolic blood pressure was more negative (P < 0.001) during posthandgrip muscle ischemia (-201.9 +/- 20.4 units. beat(-1). mmHg(-1)) when compared with control conditions (-142.7 +/- 17.3 units. beat(-1). mmHg(-1)). No significant change in the slope of the relationship between heart rate and systolic blood pressure was observed. However, both curves shifted during postexercise ischemia to accommodate the elevation in blood pressure and MSNA that occurs with this condition. These data suggest that the sensitivity of baroreflex modulation of MSNA is elevated by muscle metaboreceptor stimulation, whereas the sensitivity of baroreflex of modulate heart rate is unchanged during posthandgrip muscle ischemia.

Membrane specificity of non-monotonic trends in cation-mediated injury to rapidly frozen phospholipid vesicles.

Phospholipid vesicles like erythrocyte ghosts [1] have been shown to display trends in freeze-thaw injury with Group I ions which are non-monotonic in nature. That is to say, the relative extent of injury with such ions, measured as calcein release, does not follow a lyotropic series related solely to the hydrated ionic radii of these ions. By incorporation of cholesterol into the vesicles the non-monotonic nature of these trends has been shown to be highly membrane specific. Thus the non-monotonic trends in cation mediated freeze-thaw injury are shown to be independent of bulk solution properties.

Temperature-dependent effect of edetate disodium on neomycin stability.

When heated at temperatures in excess of 100 degrees, the stability of neomycin in aqueous ophthalmic formulations was improved by the addition of edetate disodium (0.01%). As the exposure temperature was reduced, the degree of stability enhancement diminished until the effect was reversed, and addition of edetate disodium was detrimental to neomycin stability in solutions stored at 30 degrees.

A comparison of chemically defined and complex media for the production of Bacillus subtilis spores having reproducible resistance and germination characteristics.

Spores of Bacillus subtilis were produced on five batches of Antibiotic Assay Medium No. 1 obtained from different suppliers, five batches obtained from a single supplier and five batches of a chemically defined liquid medium. The magnitude of the variation within each of the three groups was compared in terms of heat resistance, glutaraldehyde resistance, germination rate and sensitivity of germinated spores to neomycin. For all these parameters the degree of reproducibility achieved by using a chemically defined liquid medium was substantially better than that using batches of complex medium from a single supplier. Even larger variations in these parameters resulted when different suppliers were used. The value of defined media for the production of spore inocula to be used in sterilization control and similar procedures is discussed.

A comparative investigation of glycinebetaine and dimethylsulphoxide as liposome cryoprotectants.

The release of streptomycin from lecithin liposomes following a freeze-thaw cycle was used to measure the cryoprotective activities of glycinebetaine and dimethylsulphoxide (DMSO). At concentrations between 4 and 8% w/v in the external solution, glycinebetaine was superior to DMSO at freezing rates faster than 50 degrees C min-1. At lower rates their activities were similar, and drug loss ranged between 10 and 20% depending upon freezing rate and cryoprotectant concentration. The pattern of streptomycin loss when the concentrations of cryoprotectants inside and outside the liposome were varied indicated that glycinebetaine, in contrast to DMSO, does not diffuse across the liposome membrane. The activity of glycinebetaine was not impaired by the presence in the membrane of cholesterol or charged lipids.

Factors influencing cryoprotective activity and drug leakage from liposomes after freezing.

The effects of freezing and thawing conditions and cryoprotective additives on release of streptomycin from lecithin liposomes following freeze-thaw cycles have been investigated. Drug retention was maximized by slow cooling (approx. 1 degree C min-1). At temperatures between 0 degree and -20 degrees C, the extent of drug loss was time-dependent indicating incomplete freezing; below -45 degrees C this effect was abolished and the system was stable. Osmotic gradients across the liposome membrane during freezing were found to have little effect on drug loss. Marked cryoprotective activity was shown by dimethylsulphoxide, glycerol, alanine and glycinebetaine at concentrations of 3% w/v or less. At this concentration sucrose and mannitol had little activity.

The retention of entrapped molecules within erythrocyte ghosts during cryopreservation.

In view of the interest in erythrocyte ghosts and carrier erythrocytes as potential drug delivery systems, this work was undertaken to determine conditions facilitating the retention of entrapped molecules during cryopreservation. Upon freeze-thaw treatment intact erythrocytes and erythrocyte ghosts displayed different damage profiles with respect to cryoprotectant concentration. Non-penetrating cryoprotectants showed optimum protection of intact cells at 0.4-0.5 M; this optimum was not observed with ghosts, in which damage decreased with concentration up to 1.0 M. The concentration optimum for intact cells was not abolished by oxidative or reductive treatments suggesting that its absence in ghosts is not due to altered protein-protein or protein-lipid interactions. The extent of freeze-thaw damage to ghosts was influenced by the qualitative ionic composition of a cryoprotectant-free suspending medium, with 10-12% haemolysis observed in the presence of Li+ and Mg2+ but greater than 60% for Na+, Cs+, K+ and NH4+ with increasing loss following that order. The release on freezing of entrapped haemoglobin, insulin and sucrose was found to be inversely proportional to their molecular weights.

Preservative efficacy tests in formulated nasal products: reproducibility and factors affecting preservative activity.

Preservative efficacy tests were performed in triplicate on each of three batches of three formulated nasal spray preparations to assess the inter- and intra-batch variation in preservative performance which typically results from these procedures, and to assess the relative importance of factors influencing preservative performance in nasal products. Tests were conducted using procedures conforming, as far as possible, to both the European and the US pharmacopoeias and the results interpreted using the performance criteria of both. Despite the adoption of practices designed to maximize reproducibility, a marked variation in the degree of microbial inactivation was observed, both within and between batches of product. A preservative system comprising benzalkonium chloride and phenylethyl alcohol was found to be far superior to combinations of either benzalkonium chloride plus disodium edetate or potassium sorbate plus disodium edetate, both of which failed to satisfy the EP performance criteria on a number of occasions. Proposals are made for the adoption of inactivation criteria which incorporate realistic error limits reflecting the inherent problems of reproducibility of the viable counting procedures involved.

Biofilm formation and changes in bacterial cell surface hydrophobicity during growth in a CAPD model system.

Peritonitis is a frequent complication of continuous ambulatory peritoneal dialysis (CAPD), with patients suffering recurrent attacks. The microorganisms most frequently implicated in the infection are the skin microflora, in particular, the coagulase-negative staphylococci such as Staphylococcus epidermidis. These microorganisms gain access to the peritoneal cavity via the in-dwelling silicone rubber catheter in the abdominal wall and often persist as biofilms on the surface of the catheter. The surface characteristics of S. epidermidis were monitored during growth in a CAPD in-vitro model together with their ability to adhere to silicone rubber substrata. Fresh dialysis fluid exerted an injurious effect on the cells leading to a decrease in cell numbers but during the simulated dialysis period the cells adapted to the applied stresses. Over a 96-h period in the model both a clinical isolate and a skin isolate of S. epidermidis adopted a more hydrophobic phenotype. The data presented here show that the bacteria grown in this in-vivo reflective CAPD model continually adapt to their environment and become more tolerant to the stresses imposed. The adapted cells were seen to colonise silicone rubber substrata.

Bacitracin and protease production in relation to sporulation during exponential growth of Bacillus licheniformis on poorly utilized carbon and nitrogen sources.

Growth of Bacillus licheniformis in a chemically defined medium containing glucose and ammonium chloride yielded a doubling time of 1.00 h. Examination of the culture during exponential growth revealed a lack of heat-resistant spores together with a complete absence of detectable concentrations of bacitracin or extracellular serine protease. Replacement of glucose as the sole carbon source by glycerol, pyruvic acid, citric acid, or lactic acid resulted in doubling times of 1.13, 2.00, 3.16, and 3.95 h, respectively. Bacitracin, protease, and heat-resistant spores were produced during exponential growth in amounts related to these doubling times. A qualitatively similar pattern was observed when ammonium chloride was replaced by sodium nitrate, alanine, or glutamic acid which gave doubling times of 1.65, 1.77, and 1.90 h, respectively. Protease, but not bacitracin, concentrations were substantially higher when the growth rate was restricted by use of poor nitrogen rather than poor carbon sources. The relationships between bacitracin production, protease production, and the sporulation process are discussed.

Protection of Pseudomonas aeruginosa against ciprofloxacin and beta-lactams by homologous alginate.

Pseudomonas aeruginosa-derived alginate but no other neutral and negatively charged polysaccharides protected mucoid and nonmucoid strains of that organism against ciprofloxacin, gentamicin, ticarcillin, and ceftazidime. Data indicate that alginate has an intrinsic protective effect which is independent of diffusion, charge, or biofilm phenomena.

Antibiotic interaction and diffusion through alginate and exopolysaccharide of cystic fibrosis-derived Pseudomonas aeruginosa.

The interaction of five anti-pseudomonas antibiotics with both commercial and pseudomonas alginates was studied by investigation of their binding and diffusion characteristics. The two sources of alginate were qualitatively but not quantitatively similar in these respects. Unlike the beta-lactams, gentamicin and tobramycin bound avidly to both sources of alginate and, when the alginate gel to antibiotic ratio was high, the aminoglycosides exhibited diffusion coefficients which were approximately 20% of the beta-lactam values. At much lower ratios of alginate to antibiotic the aminoglycosides caused precipitation in the alginate with apparent disruption of the gel structure, and ultimately penetrated the gel at a faster rate than the beta-lactams. The strong aminoglycoside binding to alginate was reduced, but not eliminated by the presence of physiological concentrations of salts.

The diffusion of beta-lactam antibiotics through mixed gels of cystic fibrosis-derived mucin and Pseudomonas aeruginosa alginate.

The rates of diffusion through purified extracellular alginate from Pseudomonas aeruginosa were measured for twelve beta-lactam antibiotics. The diffusion rate was reduced as the antibiotic molecular weight increased, but the range of diffusion rates exhibited by a common anti-pseudomonal penicillins was relatively small. The diffusion of ticarcillin through 1.0% w/v mixtures of alginate and purified mucus glycoprotein (mucin) from sputa of cystic fibrosis patients showed that, at equivalent concentrations, alginate represented the greater barrier to penetration. However if the mucin concentration was increased to 4.0% w/v, a more realistic physiological concentration, the diffusion of ticarcillin was retarded to a greater extent than in 1% w/v alginate, and the effect was compounded by other sputum components such as DNA. The results suggest that the antibiotic diffusion barrier represented by mucin may be significant in vitro, particularly for nebulized antibiotics.

Use of slime dispersants to promote antibiotic penetration through the extracellular polysaccharide of mucoid Pseudomonas aeruginosa.

Agents with the potential to reduce Pseudomonas aeruginosa alginate viscosity (slime dispersants) were shown to promote the diffusion of antipseudomonal antibiotics through alginate but were more effective in facilitating the diffusion of gentamicin than that of ceftazidime. EDTA increased the diffusion rates of these antibiotics by factors of 4.0 and 1.5, respectively, although sodium chloride significantly reduced viscosity and enhanced gentamicin diffusion.

The influence of glucose, ammonium and magnesium availability on the production of protease and bacitracin by Bacillus licheniformis.

Bacillus licheniformis was cultivated in a range of defined media varying in both the nature of the growth-limiting component and the concentration of excess nutrients. The compositions of the media were such as to ensure that the final absorbance (A430) of the culture was the same in each case. Samples taken during the stationary phase were assayed for their content of extracellular serine protease and bacitracin. The nature of the growth-limiting nutrient had a profound effect on the amounts of these products formed while those components which were present in excess also exerted an influence in proportion to their concentration. Thus, for example, a four-fold increase in serine protease production occurred when ammonium replaced glucose as the growth-limiting nutrient. Serine protease and bacitracin production responded differently to these varying cultural conditions suggesting they are subject to separate control mechanisms. The results are discussed in relation to the need for rigorously controlled cultural conditions during physiological studies of this nature.

Nonmonotonic trends in electrolyte-induced injury to rapidly cooled erythrocytes.

Rapid freeze-thaw injury to erythrocytes and erythrocyte ghosts has been shown to be strongly cation dependent. For the Group I ions this dependence is nonmonotonic in nature with injury increasing in the order Li+ less than Na+ less than Cs+ less than K+. Injury can be reduced by the inclusion in the freezing media of saccharide cryoprotectants or by the substitution with less injurious cations, e.g., Mg2+ or (CH3)4N+. In contrast to the situation observed with cations injury with anions follows Hofmeister lyotropic power series with injury increasing with decreasing hydrated ionic radius. Careful choice of electrolyte species allows injury to be reduced to levels comparable to that afforded by saccharide cryoprotectants. A possible mechanism for the nonmonotonic trends in injury observed with cations is considered.