Coordinated regulation of cold-induced changes in fatty acids with cardiolipin and phosphatidylglycerol composition among phospholipid species for the food pathogen Listeria monocytogenes.

We present here the structural identification of four phospholipid (Phl) classes in Listeria monocytogenes, the fatty acid (FA) composition for each individual Phl species, and a description of cold-induced FA changes. Cardiolipin (48.5%) and phosphatidylglycerol (18.1%) are dominated by anteiso-FA, and the previously recognized branched FA chain shortening by cold was observed singularly in these Phls. Phosploaminolipid (19.9%) and phosphatidylinositol, (9.1%) are significantly different, containing significant amounts of straight-chain FA. These findings are supported by nuclear magnetic resonance analysis.

Effect of cold temperature on the composition of different lipid classes of the foodborne pathogen Listeria monocytogenes: focus on neutral lipids.

In this work a thorough consideration of the membrane lipid composition of Listeria monocytogenes together with DSC analysis is described in order to estimate the biological importance of lipid changes during low-temperature adaptation. Furthermore, these studies provide comparative data for fatty acid changes for neutral, NL and polar lipids, PL separately. The cold adaptation (5 degrees C) response of L. monocytogenes showed (i) an increase in the level of NL content (30%) among the total lipids, TL and (ii) that the increase (7-fold) in the anteiso-15:0/anteiso-17:0 fatty acid ratio, FAr, for cold NL was at variance with the ratio for TL and PL (about 10-fold). We correlated our findings with DSC studies on phase transition temperature (Tc), enthalpy difference (DeltaH) and peak range of the transition for TL, PL, NL (from cultures at 30 and 5 degrees C); The decrease of Tc (10.5 degrees C) and DeltaH (51%) for TL is a reflection of the decrease of Tc (11.5 degrees C) and DeltaH (56%) for PL. This large decrease is interpreted by the high (10-fold) increase of a-15:0/a-17:0 FAr of PL5 degrees C. In NL the decrease of Tc (3 degrees C) and of DeltaH (42%) is interpreted by both adaptation mechanisms: the (lower) 7-fold increase of anteiso-15:0/anteiso-17:0 FAr and the NL percentage calculated from increased mass values. The peak range of TL5 degrees C (from -15 to 25 degrees C) is a reflection of the peak range of NL5 degrees C, which is unchanged, as is the peak range of NL30 degrees C.

Residual blood loss in single use dialyzers: effect of different membranes and flux.

AIM/BACKGROUND: The aim of this study was to evaluate the residual blood loss in new type single use dialyzers under the usually prevailing conditions during hemodialysis and to investigate whether or not this loss is dependent on dialyzer membrane composition or flux characteristics. MATERIALS AND METHODS: In 158 hemodialysis (HD) patients, 158 single used dialyzers were studied in corresponding HD sessions. 52/158 dialyzers were made from modified cellulose (acetate, CA or triacetate, CTA) membrane and 106 from synthetic ones (58 with ethyl-vinyl-alcohol (EVAL), 48 with polyacrilonitrile (AN69)). Of those dialyzers 85/158 (58 EVAL+27CA) were low flux (LF) while the other 73 were high flux (HF). Patients underwent 4 hour HD sessions and at the end of the session blood was drawn for the measurement of hematocrit (Ht) and hemoglobin (Hb). Additionally, after the end of dialysis the used dialyzers were rinsed with 1000 mL of 0.05% NH(3) solution in distilled water. The wash was collected and subsequently Hb was measured using the benzidine method. From the volume of the solution and its concentration of Hb, total Hb of the solution was measured and blood loss in terms of red blood cell (RBC) volume was estimated by the use of the formula: RBC (mL) = Total Hb (g) in the solution x patient's Ht (ml/dL) / Patient's Hb (g/dL). For results to be comparable between dialyzers, RBC volume/m(2) of dialyzer membrane was expressed. In 5/158 patients blood loss was also estimated in 6 consecutive HD sessions using the same type of dialyzer. RESULTS: For the sum of the dialyzers, blood loss / dialyzer in terms of RBC volume, expressed as median (range), was 0.978 mL (0.01-23.9). There was statistically significant (p<0.001 or p<0.05) higher blood loss with the use of AN69 dialyzer than with the other three. RBC HF >RBC LF (p<0.001) constrained the first group of patients to use a 6% higher dosage of ferrum and 3.5% higher dosage of erythropoietin than the other group to achieve the optimal hemoglobulin values. No difference existed regarding RBC between CA, CTA and EVAL membranes. RBC measured in the small group of 5/158 patients for 6 consecutive HD sessions with the same dialyzer showed a wide range of RBC loss indicating an effect of the human factor. CONCLUSIONS: Blood loss during HD sessions due to residual blood cell volume inside dialyzers is usually slight using new type single use dialyzers but, sometimes, it can be significant and may contribute to the development or deterioration of preexisting iron deficiency anemia. The results of this study indicated that this loss can be attributed to the membrane composition of the dialyzer or to the human factor and has nothing to do with the ultrafiltration coefficient of the dialyzer.

Characterization and replication properties of the Zymomonas mobilis ATCC 10988 plasmids pZMO1 and pZMO2.

The complete nucleotide sequences of two small cryptic Zymomonas mobilis ATCC 10988 plasmids (pZMO1 and pZMO2) were determined. The plasmids showed 67% homology to each other at their nucleotide level. Plasmid pZMO1 was 1651 bp long with 38% G + C content and contained an open reading frame (ORFZMO1) of 1044 nucleotides. ORFZMO1 is predicted to encode a polypeptide of 348 amino acids and shows a high degree of homology with gram-negative replication proteins of rolling circle replicating plasmids, which belong to the pC194/pUB110 family. Plasmid pZMO2 was found to be 1669 bp long, with a 38.5% G + C content, and it contained an ORF of 552 nucleotides (ORFZMO2) encoding a putative polypeptide of 184 amino acids. This polypeptide also shows a high degree of homology with the replication proteins of RCR plasmids of gram-negative bacteria, but only at their N-termini. The region necessary for replication of both plasmids was determined by stability tests under nonselective conditions, following cloning in pBR325 and introduction in Z. mobilis ATCC 10988 by pRK2013 assisted conjugation. Double- and single-strand origin regions were predicted by sequence analysis. Detection of single-stranded DNA in the extract of exponentially growing cells confirmed experimentally the rolling circle replication mode of at least pZMO2.

Characterization of the mobilization region of the Zymomonas mobilis ATCC10988 plasmid pZMO3.

The 2.7-kb Zymomonas mobilis ATCC10988 plasmid pZMO3 contains a coding region (ORF1) indispensable for mobilization. A cis-acting 409-bp sequence between ORF2 (C-terminal) and ORF1 (N-terminal) conferred mobilization activity to pUC19, when the product of ORF1 was provided in trans. In this area, two segments showed homology with previously characterized oriT regions.

Development of a gene reporter system in moderately halophilic bacteria by employing the ice nucleation gene of Pseudomonas syringae.

The expression of the ice nucleation gene inaZ of Pseudomonas syringae in several moderate halophiles was investigated to establish its utility as a reporter for promoter activity and gene expression studies in these biotechnologically and environmentally important bacteria. A promoterless version of inaZ was introduced in two different restriction sites and at both orientations in a recombinant plasmid able to replicate in moderate halophiles and, in particular, within the sequence of its pHE1 part, a native plasmid of Halomonas elongata. One orientation of both recombinant constructs expressed high levels of ice nucleation activity in H. elongata and Volcaniella eurihalina cells, indicating that inaZ was probably introduced in the correct orientation downstream of putative native promoters. A recombinant construct carrying a tandem duplication of inaZ at the same orientation gave significantly higher ice nucleation activity, showing that inaZ is appropriate for gene dosage studies. The ice nucleation gene was also expressed in H. elongata and V. eurihalina under the control of Pbla (the promoter of the beta-lactamase gene of Escherichia coli) and Ppdc (the promoter of the pyruvate decarboxylase gene of Zymomonas mobilis). One of the inaZ reporter plasmids expressing high levels of ice nucleation activity under the control of a native putative promoter was also transferred in Halomonas subglaciescola, Halomonas meridiana, Halomonas halodurans, and Deleya halophila. In all cases, Ice+ transconjugants were successfully isolated, demonstrating that inaZ is expressed in a wide spectrum of moderately halophilic species.