Isolation and Characterization of Alicycliphilus denitrificans Strain BC, Which Grows on Benzene with Chlorate as the Electron Acceptor

A bacterium, strain BC, was isolated from a benzene-degrading chlorate-reducing enrichment culture. Strain BC degrades benzene in conjunction with chlorate reduction. Cells of strain BC are short rods that are 0.6 μm wide and 1 to 2 μm long, are motile, and stain gram negative. Strain BC grows on benzene and some other aromatic compounds with oxygen or in the absence of oxygen with chlorate as the electron acceptor. Strain BC is a denitrifying bacterium, but it is not able to grow on benzene with nitrate. The closest cultured relative is Alicycliphilus denitrificans type strain K601, a cyclohexanol-degrading nitrate-reducing betaproteobacterium. Chlorate reductase (0.4 U/mg protein) and chlorite dismutase (5.7 U/mg protein) activities in cell extracts of strain BC were determined. Gene sequences encoding a known chlorite dismutase (cld) were not detected in strain BC by using the PCR primers described in previous studies. As physiological and biochemical data indicated that there was oxygenation of benzene during growth with chlorate, a strategy was developed to detect genes encoding monooxygenase and dioxygenase enzymes potentially involved in benzene degradation in strain BC. Using primer sets designed to amplify members of distinct evolutionary branches in the catabolic families involved in benzene biodegradation, two oxygenase genes putatively encoding the enzymes performing the initial successive monooxygenations (BC-BMOa) and the cleavage of catechol (BC-C23O) were detected. Our findings suggest that oxygen formed by dismutation of chlorite can be used to attack organic molecules by means of oxygenases, as exemplified with benzene. Thus, aerobic pathways can be employed under conditions in which no external oxygen is supplied.     

Pharmacokinetics of poly(hydroxyethyl-l-asparagine)-coated liposomes is superior over that of PEG-coated liposomes at low lipid dose and upon repeated administration

‘Stealth’ liposomes with a poly(ethylene glycol) (PEG) coating are frequently studied for drug delivery and diagnostic purposes because of their prolonged blood circulation kinetics. However, several recent reports have demonstrated that PEG-liposomes are rapidly cleared at single low lipid doses (< 1 μmol/kg) and upon repeated administration (time interval between the injections 5 days-4 weeks). Recently, poly(amino acid)-based stealth liposome coatings have been developed as alternative to the PEG-coating. In this study, the pharmacokinetic behavior of liposomes coated with the poly(amino acid) poly(hydroxyethyl-l-asparagine) (PHEA) was evaluated at low lipid doses and upon repeated administration in rats. Blood circulation times and hepatosplenic localization of PHEA-liposomes were assessed after intravenous injection. When administered at a dose of 0.25 μmol/kg or less, PHEA-liposomes showed significantly longer blood circulation times than PEG-liposomes. A second dose of PHEA-liposomes 1 week after the first injection was less rapidly cleared from the circulation than a second dose of PEG-liposomes. Although the mechanisms behind these observations are still not clear yet, the use of PHEA-liposomes appears beneficial when single low lipid doses and/or repeated dosing schedules are being applied.     

Improved identification of heterozygotes for homocystinuria due to cystathionine synthase deficiency by the combination of methionine loading and enzyme determination in cultured fibroblasts

Summary Previous data on tentative identification of the carrier state for homocystinuria due to cystathionine synthase deficiency using methionine loading or measurement of cystathionine synthase activity in tissue extracts are conflicting. We studied the results of standardized oral methionine loading in 20 obligate heterozygotes and compared them with those of determination of cystathionine synthase activity in cultured fibroblasts. Special attention was devoted to our recently reported observation on the small but striking differences in methionine metabolism between healthy pre- and postmenopausal women and men. Fasting and after load peak levels of methionine in serum did not discriminate the carriers from the control subjects. The mean fasting level of total homocysteine was only significantly higher in the group of premenopausal heterozygotes than in the corresponding control group. Nevertheless, the individual values overlapped with the normal range in 4 of 12 premenopausal heterozygotes. After loading peak levels of total homocysteine in 18 out of the 20 obligate heterozygotes exceeded the upper limit of the ranges in the three control groups. Thus, this parameter discriminated 90% of the obligate carriers. Measurement of cystathionine synthase activity in cultured fibroblasts from a skin biopsy identified the obligate heterozygotes to a similar degree (85%). No significant correlation between the measurements of cystathionine synthase activity and the after load peak levels of total homocysteine in the individual heterozygotes was established. Combination of both methionine loading and determination of cystathionine synthase activity in cultured fibroblasts identified all of these carriers.     

Ultrastructural effects of antimicrobial peptides from bovine lactoferrin on the membranes of Candida albicans and Escherichia coli

Antimicrobial peptides allegedly exert their action on microbial membranes. Bovine lactoferrin enfold two antimicrobial domains, lactoferricin B (LFcin B) and lactoferrampin (LFampin). Effects of representative peptides thereof on the membranes of Candida albicans and Escherichia coli were investigated. Confocal laser scanning microscopy revealed that these peptides were internalized within a few minutes, concurrently with disrupting membrane integrity as indicated by freeze-fracture transmission electron microscopy. The most striking findings were induction of distinct vesicle-like structures in the membrane of C. albicans by the LFampin peptide, and detachment of the outer membrane and surface protrusions in E. coli by the LFcin B peptide.     

Incomplete quality of life data in lung transplant research: comparing cross sectional,repeated measures ANOVA,and multi-level analysis

Background

In longitudinal studies on Health Related Quality of Life (HRQL) it frequently occurs that patients have one or more missing forms, which may cause bias, and reduce the sample size. Aims of the present study were to address the problem of missing data in the field of lung transplantation (LgTX) and HRQL, to compare results obtained with different methods of analysis, and to show the value of each type of statistical method used to summarize data.

Methods

Results from cross-sectional analysis, repeated measures on complete cases (ANOVA), and a multi-level analysis were compared. The scores on the dimension ''energy'' of the Nottingham Health Profile (NHP) after transplantation were used to illustrate the differences between methods.

Results

Compared to repeated measures ANOVA, the cross-sectional and multi-level analysis included more patients, and allowed for a longer period of follow-up. In contrast to the cross sectional analyses, in the complete case analysis, and the multi-level analysis, the correlation between different time points was taken into account. Patterns over time of the three methods were comparable. In general, results from repeated measures ANOVA showed the most favorable energy scores, and results from the multi-level analysis the least favorable. Due to the separate subgroups per time point in the cross-sectional analysis, and the relatively small number of patients in the repeated measures ANOVA, inclusion of predictors was only possible in the multi-level analysis.

Conclusion

Results obtained with the various methods of analysis differed, indicating some reduction of bias took place. Multi-level analysis is a useful approach to study changes over time in a data set where missing data, to reduce bias, make efficient use of available data, and to include predictors, in studies concerning the effects of LgTX on HRQL.     

Evolution of dorsal-ventral axis formation in arthropod appendages: H15 and optomotor-blind/bifid-type T-box genes in the millipede Glomeris marginata (Myriapoda: Diplopoda)

In Drosophila, the T-box genes optomotor-blind (omb) and H15 have been implicated in specifying the development of the dorso-ventral (DV) axis of the appendages. Results from the spider Cupiennius salei have suggested that this DV patterning system may be at least partially conserved. Here we extend the study of the DV patterning genes omb and H15 to a representative of the Myriapoda in order to add to the existing comparative data set and to gain further insight into the evolution of the DV patterning system in arthropod appendages. The omb gene of the millipede Glomeris marginata is expressed on the dorsal side of all appendages including trunk legs, maxillae, mandibles, and antennae. This is similar to what is known from Drosophila and Cupiennius and suggests that the role of omb in instructing dorsal fates is conserved in arthropods. Interestingly, the lobe-shaped portions of the mouthparts do not express omb, indicating that these are ventral components and thus may be homologous to the endites present in the corresponding appendages in insects. Concerning the H15 gene we were able to identify two paralogous genes in Glomeris. Both genes are expressed in the sensory organs of the maxilla and antenna, but only Gm-H15-1 is expressed along the ventral side of the trunk legs. The expression is more extensive than in Cupiennius, but less so than in Drosophila. In addition, no ventral expression domain is present in the maxilla, mandible, and antenna. Because of this, the role of H15 in the determination of ventral fate remains unclear.     

Pair rule gene orthologs in spider segmentation

The activation of pair rule genes is the first indication of the metameric organization of the Drosophila embryo and thus forms a key step in the segmentation process. There are two classes of pair rule genes in Drosophila: the primary pair rule genes that are directly activated by the maternal and gap genes and the secondary pair rule genes that rely on input from the primary pair rule genes. Here we analyze orthologs of Drosophila primary and secondary pair rule orthologs in the spider Cupiennius salei. The expression patterns of the spider pair rule gene orthologs can be subdivided in three groups: even-skipped and runt-1 expression is in stripes that start at the posterior end of the growth zone and their expression ends before the stripes reach the anterior end of the growth zone, while hairy and pairberry-3 stripes also start at the posterior end, but do not cease in the anterior growth zone. Stripes of odd-paired, odd-skipped-related-1, and sloppy paired are only found in the anterior portion of the growth zone. The various genes thus seem to be active during different phases of segment specification. It is notable that the spider orthologs of the Drosophila primary pair rule genes are active more posterior in the growth zone and thus during earlier phases of segment specification than most orthologs of Drosophila secondary pair rule genes, indicating that parts of the hierarchy might be conserved between flies and spiders. The spider ortholog of the Drosophila pair rule gene fushi tarazu is not expressed in the growth zone, but is expressed in a Hox-like fashion. The segmentation function of fushi tarazu thus appears to be a newly acquired role of the gene in the lineage of the mandibulate arthropods.     

Temperature- and Light-Dependent Performance of the Cyanobacterium <Emphasis Type="Italic">Leptolyngbya Foveolarum</Emphasis> and the Diatom <Emphasis Type="Italic">Nitzschia Perminuta</Emphasis> in Mixed Biofilms

This paper investigates the role of species interactions as a mechanism determining the changing seasonal abundance of microphytobenthic species. Different kinds of interactions can occur between microphytobenthic species, e.g., interference competition (a species directly hindering the growth of another) or resource competition. If such interactions are strong, the capacity of species to exploit parts of the seasonal spectrum of temperature and light conditions could be greatly affected. A model system of two freshwater benthic phototrophs, the cyanobacterium Leptolyngbya foveolarum (Rabenhorst ex Gomont) Anagnostidis et Komárek and the diatom Nitzschia perminuta (Grunow) M. Pergallo, was used to study the capacity of each species to grow in ranges of temperature (7, 15 and 25 °C) and light (5, 40 and 200 μmol m⁻² s⁻¹) conditions in single-species and two-species cultures. Growth was followed for 14–17 days by measuring chlorophyll a and maximum photosynthetic capacity using spectrophotometry and pulse amplitude modulated (PAM) fluorimetry. A PHYTO-PAM fluorimeter facilitated simultaneous observations in mixed cultures on the two species. In mixed cultures, the diatom appeared to be a ‘cool season species’ (low temperature and low light intensity) and the cyanobacterium a ‘summer or autumn species’ (higher temperature and light intensities). This is different than predicted by monoculture experiments, where a wide range of optimal growth conditions was found. Two-species biofilm tests indicated inhibitory effects of the cyanobacterium on the diatom species, especially under conditions favorable to the cyanobacterium. High or low light intensities, increase of local pH caused by depletion of inorganic carbon, and limitation of other inorganic nutrients (resource competition) were examined as factors contributing to diatom inhibition, but none provided an acceptable explanation for observed growth patterns. Our results pointed towards interference competition.