Immobilization of lipoxygenase in an alginate-silicate solgel matrix: formation of fatty acid hydroperoxides

A method for the immobilization of lipoxygenase (LOX) in an alginate-silicate gel matrix was developed. In this method, a mixture of calcium alginate beads and LOX in borate buffer are dispersed into a hexane solution of tetramethoxy-ortho-silicate (TMOS). Hydrolysis of the TMOS gives products that permeate and co-polymerize with the alginate gel to form a colloid within the beads that entraps the LOX. Optimum reaction conditions for sol-gel entrapment of LOX are at pH 9.0 in 0.2M borate buffer. The composite gel, after isolation and vacuum drying, had excellent protein retention that has good enzyme activity and stability at room temperature. The activity of the entrapped LOX was less than the activity of the free enzyme. However, the activity of the immobilized LOX can be restored by the addition of borate buffer and glycerol, or borate buffer saturated with an organic solvent. In contrast to the free enzyme in solution, which loses its activity in less than one day, sol-gel entrapped LOX retains its activity at ambient temperature for at least 25 days and can be recycled. This report demonstrates that the sol-gel entrapment method for immobilizing LOX can be useful in developing a process for the oxidation of polyunsaturated fatty acids.     

Replicate plating: does it increase reliability?

K.K. WILLE, B.R. VOWELS, A.N. FOGLIA, C.A. BERGE, B.M. SCHNELL AND F.W. BRIESE. 1996. As a part of a clinical study to evaluate the antibacterial effect of a topically applied erythromycin gel, microbiological specimens were taken from two groups of patients : one group using 2% erythromycin gel and the other group using a placebo gel. These specimens were plated in triplicate using a common source on bacteriological media using standard procedures. After the appropriate incubation times, the numbers of aerobic and anaerobic organisms were counted separately from each of three plates. A comparison of the bacterial colony counts from the replicate plates showed a high degree of similarity for each type of organism. Tests for treatment differences in organism counts were performed based on single, double and triplicate plating. The results obtained were almost identical, suggesting that replicate plating from a common source is no more accurate than single plating. The only apparent advantage of this type of replicate plating is heightened confidence in the reliability of bacterial counts from single plates.     

Improved method for extraction of hydroperoxide lyase from Chorella

Summary Hydroperoxide lyase converts fatty acid hydroperoxides to oxo-fatty acids which, after further oxidation, are suitable for the synthesis of higher polyamides. An improved method utilizing an acetone powder step for isolation of this enzyme from the unicellular alga Chlorella was developed. Using this procedure a five fold increase in hydroperoxide lyase activity from Chlorella pyrenoidosa was obtained compared to previously used extraction methods. Other Chlorella species were assayed, and it was found that C. fusca also contained significant hydroperoxide lyase activity.     

Cost-Utility Analysis of Lopinavir/Ritonavir versus Atazanavir + Ritonavir Administered as First-Line Therapy for the Treatment of HIV Infection in Italy: From Randomised Trial to Real World

Objective

To estimate the lifetime cost utility of two antiretroviral regimens (once-daily atazanavir plus ritonavir [ATV+r] versus twice-daily lopinavir/ritonavir [LPV/r]) in Italian human immunodeficiency virus (HIV)-infected patients naïve to treatment.

Design

With this observational retrospective study we collected the clinical data of a cohort of HIV-infected patients receiving first-line treatment with LPV/r or ATV+r.

Methodology

A Markov microsimulation model including direct costs and health outcomes of first- and second-line highly active retroviral therapy was developed from a third-party (Italian National Healthcare Service) payer’s perspective. Health and monetary outcomes associated with the long-term use of ATV+r and LPV/r regimens were evaluated on the basis of eight health states, incidence of diarrhoea and hyperbilirubinemia, AIDS events, opportunistic infections, coronary heart disease events and, for the first time in an economic evaluation, chronic kidney disease (CKD) events. In order to account for possible deviations between real-life data and randomised controlled trial results, a second control arm (ATV+r 2) was created with differential transition probabilities taken from the literature.

Results

The average survival was 24.061 years for patients receiving LPV/r, 24.081 and 24.084 for those receiving ATV+r 1 and 2 respectively. The mean quality-adjusted life-years (QALYs) were higher for the patients receiving LPV/r than those receiving ATV+r (13.322 vs. 13.060 and 13.261 for ATV+r 1 and 2). The cost-utility values were 15,310.56 for LPV/r, 15,902.99 and 15,524.85 for ATV+r 1 and 2.

Conclusions

Using real-life data, the model produced significantly different results compared with other studies. With the innovative addition of an evaluation of CKD events, the model showed a cost-utility value advantage for twice-daily LPV/r over once-daily ATV+r, thus providing evidence for its continued use in the treatment of HIV.     

Patterns of Root Colonization in Epacridaceous Plants Collected from Different Sites

Root colonization was studied in ten species of the Epacridaceaeat three sites in Victoria by morphological and cross-inoculationexperiments. The sites and genera chosen were Cranbourne [Epacrisimpressa Labill. andLeucopogon ericoides(Smith) R. Br.] andRye [L. parviflorus(Andrews) Lindley] on the Mornington Peninsula,and the Grampians[Astroloma conostephioides(Sond.) Benth.,A.humifusum(Cav.) R. Br.,A pinifolium(R. Br.) Benth,Brachylomadaphnoides(Smith) Benth.,E. impressa, E. impressavar.grandifloraBenth.andStyphelia adscendensR. Br.] in western Victoria. For morphologicalstudies, samples of roots from each species at each site werecleared and stained and examined microscopically. For cross-inoculationstudies, cuttings from each site were struck in potting mediuminoculated with soil from the same and other sites. The ericoidmycorrhizae in the roots of plants found at or grown in Cranbourneand Rye soils were similar. Both were significantly differentfrom the internal hyphae found in the roots of plants foundat or grown in Grampians soils, which were three times largerin diameter and formed dense coils which filled the host celland invaded adjacent epidermal cells. This suggests that morethan one fungus is involved in the relationships, that the MorningtonPeninsula sites had a different fungus from the Grampians siteand that host specificity is low. Vesicular structures werealso found commonly on plants at the Grampians site, in contrastwith other sites. Epacridaceae; root; fungus; mycorrhiza; morphology; inoculation     

Synthesis of poly(hydroxyalkanoates) by Escherichia coli expressing mutated and chimeric PHA synthase genes

Pseudomonas resinovorans phaC1 _Pre and phaC2 _Pre genes coding for poly(hydroxyalkanoate) (PHA) synthases were cloned by PCR and expressed in E. coli LS1298 (fadB). Repeat-unit composition analysis showed that -hydroxydecanoate (67–75 mol%) and -hydroxyoctanoate (25–33 mol%) are the major monomers of the PHA produced in cells grown on decanoate. Sequence analysis showed that the gene products of phaC1 _Pre and phaC2 _Pre had 61% identical (75% positive) amino-acid sequence matches, and both sequences contained a conserved /-hydrolase fold in the carboxy-terminal portion of the proteins. Switching the /-hydrolase folds of phaC1 _Pre and phaC2 _Pre yielded chimeric pha7 and pha8 genes that afforded PHA synthesis in E. coli LS1298. The repeat-unit compositions of PHA in cells containing pha7 and pha8 were similar to those found in transformants containing the parental genes. Deletion mutants of phaC1 _Pre and phaC2 _Pre that resulted in potential translational fusions also supported PHA synthesis with similar repeat-unit compositions. Chimeric genes obtained from the switching of fragments containing the /-hydrolase folds of phaC1 _Pre and Ralstonia eutropha phbC did not direct the synthesis of PHA in transformed cells.     

Cold shock and its effect on ribosomes and thermal tolerance in Listeria monocytogenes

Differential scanning calorimetry (DSC) and fatty acid analysis were used to determine how cold shocking reduces the thermal stability of Listeria monocytogenes. Additionally, antibiotics that can elicit production of cold or heat shock proteins were used to determine the effect of translation blockage on ribosome thermal stability. Fatty acid profiles showed no significant variations as a result of cold shock, indicating that changes in membrane fatty acids were not responsible for the cold shock-induced reduction in thermal tolerance. Following a 3-h cold shock from 37 to 0 degrees C, the maximum denaturation temperature of the 50S ribosomal subunit and 70S ribosomal particle peak was reduced from 73.4 +/- 0.1 degrees C (mean +/- standard deviation) to 72.1 +/- 0.5 degrees C (P < or = 0.05), indicating that cold shock induced instability in the associated ribosome structure. The maximum denaturation temperature of the 30S ribosomal subunit peak did not show a significant shift in temperature (from 67.5 +/- 0.4 degrees C to 66.8 +/- 0.5 degrees C) as a result of cold shock, suggesting that either 50S subunit or 70S particle sensitivity was responsible for the intact ribosome fragility. Antibiotics that elicited changes in maximum denaturation temperature in ribosomal components also elicited reductions in thermotolerance. Together, these data suggest that ribosomal changes resulting from cold shock may be responsible for the decrease in D value observed when L. monocytogenes is cold shocked.