Application of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for monitoring the digestion of phosphatidylcholine by pancreatic phospholipase A(2)

Different methods were established for monitoring the phospholipase A(2)(PLA(2)) activity but all of them are rather cumbersome and time consuming. In this paper we have investigated the suitability of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for the determination of the PLA(2) activity. Phosphatidylcholine (PC) was digested with pancreatic PLA(2) under different conditions, i.e., various Ca(2+), PC, and PLA(2) concentrations. The digestion products were analyzed by MALDI-TOF MS and the concentration of lysophosphatidylcholine (LPC)-generated upon PLA(2) digestion-was determined by the application of an internal standard (known concentration) and by a comparison of their signal-to-noise ratios. The results clearly demonstrate that the LPC concentration determined from the MALDI-TOF mass spectra correlates directly with the activity of the applied enzyme. Additionally, LPC concentration increased with an increase in Ca(2+), as well as in the PC concentration. A single MALDI-TOF mass spectrum provides immediate information on the digestion products as well as on the residual substrate without requirements for any previous derivatization. MALDI-TOF MS can be easily and simply applied for monitoring the PLA(2) activity and we assume that this method might also be useful for other types of phospholipases.     

Identification of a non-canonical tyrosine-based endocytic motif in an ionotropic receptor

Rapid modulation of the surface number of certain ionotropic receptors is achieved by altering the relative rates of insertion and internalization. These receptors are internalized by a clathrin-mediated pathway; however, a motif that is necessary for endocytosis of ionotropic receptors has not yet been identified. Here, we identified a motif that is required for constitutive and agonist-regulated internalization of the ionotropic P2X(4) receptor. Three amino acids in the C terminus of P2X(4) (Tyr(378), Gly(381), and Leu(382)) compose a non-canonical tyrosine-based sorting signal of the form YXXGL. We found that P2X(4) protein was present in clathrin-coated vesicles isolated from rat brain and that a glutathione S-transferase fusion of the P2X(4) C terminus pulled down the adaptor protein-2 complex from brain extract. Mutation of either the tyrosine-binding pocket of the mu2 subunit of adaptor protein-2 or the YXXGL motif in the receptor C terminus caused a decrease in receptor internalization and a dramatic increase in the surface expression of P2X(4) receptors. The YXXGL motif represents a non-canonical tyrosine-based sorting signal that is necessary for efficient endocytosis of the P2X(4) receptor. Similar motifs are present in other receptors and may be important for the control of their functional expression.     

Sequence of PRAT Satellite DNA ``Frozen' in Some Coleopteran Species

The intriguing diversity of highly abundant satellite repeats found even among closely related species can result from processes leading to dramatic changes in copy number of a particular sequence in the genome and not from rapid accumulation of mutations. To test this hypothesis, we investigated the distribution of the PRAT satellite DNA family, a highly abundant major satellite in the coleopteran species Palorus ratzeburgii, in eight species belonging to the related genera (Tribolium, Tenebrio, Latheticus), the subfamily (Pimeliinae), and the family (Chrysomelidae). Dot blot analysis and PCR assay followed by Southern hybridization revealed that the PRAT satellite, in the form of low-copy number repeats, was present in all tested species. The PRAT satellite detected in the species Pimelia elevata has been sequenced, and compared with previously cloned PRAT monomers from Palorus ratzeburgii and Palorus subdepressus. Although the two Palorus species diverged at least 7 Myr ago, and the subfamily Pimeliinae separated from the genus Palorus 50–60 Myr ago, all PRAT clones exhibit high mutual homology, with average variability relative to the common consensus sequence of 1.3%. The presence of ancestral mutations found in PRAT clones from all three species as well as the absence of species diagnostic mutations illustrate extremely slow sequence evolution. This unexpectedly high conservation of PRAT satellite DNA sequence might be induced by a small bias of turnover mechanisms favoring the ancestral sequence in the process of molecular drive.     

Yeast-like fungi as etiologic agents of blood infections in patients hospitalized in 1998-1999

The aim of the study was the analysis of frequency of yeast-like fungi as etiological agents of fungemias in patients hospitalized in operative and conservative wards of Medical Academy Central Clinical Hospital in Warsaw in 1998-1999. Peripheral blood samples and collected from vascular catheters were incubated in BacT/Alert system(Organon Teknika, USA). Positive blood samples were inoculated on Sabouraud medium with chloramphenicol (bioMerieux, France) (the time of cultivation from 48 h to 7 days at 30 C) and on chromogenic medium BBL CHROMagar Candida (Becton Dickinson, USA). Fungal strains were identified by standard mycological procedures using ID 32 C strips (ATB system, bioMerieux, France) and tests of Sanofi Diagnostics Pasteur (France). The total number of positive blood cultures was 1724. Fifty eight fungal strains were isolated from blood samples (3.36%). Strains belonged to 4 genera: Candida (55), Trichosporon (1), Saccharomyces (1) and Pichia (1). Thirty eight fungal strains were isolated from peripheral blood samples. Forty seven fungal strains were cultured from patients hospitalized in operative wards. Among fungi isolated from peripheral blood samples C. albicans (10), C. glabrata (9) and C. parapsilosis (5) strains dominated. From blood samples collected from vascular catheters most often C. albicans (7), C. glabrata (4) and C. parapsilosis (3) were isolated.     

Influence of calcium hydroxide root-canal sealer on microbial growth in vitro

The calcium hydroxide-based filling material Apexit, which is often used in endodontic practice, was evaluated for its antibacterial and antifungal effects against microorganisms isolated from oral cavity (Serratia marcescens, Pseudomonas putida, Staphylococcus aureus, Candida albicans). Two different quantitative techniques were employed—the direct-contact test was used to examine the efficacy of freshly mixed material while the broth-survival test was employed to check the antimicrobial properties of 5-d-old material. Apexit inhibited Gram-negative bacteria more effectively than Gram-positive ones but had none or a very weak inhibitory effect onC. albicans.     

Thyroid C cell function during fasting and refeeding of young and old rats

Age-related alterations in the structure and function of many organs often become apparent under stimulation of their function. Although the ageing process affects the regulation of mineral homeostasis, the function of thyroid C-cells that secrete calcitonin (CT) under the conditions of fasting and refeeding, a way of dietary manipulation that reveal the existence of age-related changes of follicular thyroid cells, has not been characterized. Therefore, we investigated the number of C-cells and serum CT concentration in young (4 mo) and old (26 mo) male rats fasted for 48 hours, and then refed for 4 or 24 hours. We found significantly higher number of C-cells in thyroids of old vs young rats both under basal conditions, and after fasting/refeeding. Correspondingly, serum calcitonin level was higher in fed or fasted old rats vs young ones. However, in young rats refeeding decreased, whereas in old animals increased serum concentrations of calcitonin. Thus, the control of serum calcium concentration, that was well preserved in old rats, occurs at the expense of increased serum CT level both under basal conditions, and after refeeding. These observations suggest that C-cell function is altered in ageing.     

Apolipoprotein H genetic variability in the population of Krk Island,Croatia

Apolipoprotein polymorphisms are emerging as suitable markers for the study of the formation of human populations. In contrast to the data available for apolipoprotein E, the data regarding apolipoprotein H (protein, apoH; gene, APOH) variations are only beginning to accumulate. By blood plasma isoelectric focusing and immunoblotting, we analyzed the distribution of apoH phenotypes in 397 individuals (192 males; 205 females) from seven villages of an autochthonous population of the eastern Adriatic island of Krk. APOH allele frequencies were: APOH*2 = 0.877, APOH*3 = 0.098, APOH*1 = 0.025, with the majority of the sample being homozygous. No significant differences between villages were observed. When these data were compared to those of other populations studied so far, a significant association between APOH allele frequencies and latitude was observed. We hypothesize that this association reflects differences in diet composition across different climatic zones.     

Isolation of glucocardiolipins from Geobacillus stearothermophilus NRS 2004/3a

Glucose-substituted cardiolipins account for about 4 mol% of total phospholipid extracted from exponentially grown cells of Geobacillus stearothermophilus NRS 2004/3a. Individual glucocardiolipin species exhibited differences in fatty acid substitution, with iso-C(15:0) and anteiso-C(17:0) prevailing. The compounds were purified to homogeneity by a novel protocol and precharacterized by matrix-assisted laser desorption ionization time-of-flight mass spectrometry.     

Alcohol-induced delay of viability loss in stationary-phase cultures of Escherichia coli

During prolonged incubation in stationary phase Escherichia coli undergoes starvation-induced differentiation, resulting in highly resistant cells. In rich medium with high amino acid content further incubation of cultures at high cell density leads to the generation of a population of cells no longer able to form colonies. The viability loss is due to some component of spent medium, active at high pH and high cell density, and can be prevented either by keeping the pH close to neutrality, by washing off the nonsalt components of the medium, or by keeping the saturating cell density low. Exposure to short-chain n-alcohols within a specific time window in stationary phase also prevents viability loss, in an rpoS-dependent fashion. The development of stress resistance, a hallmark of stationary-phase cells, is affected following alcohol treatment, as is the response to extracellular factors in spent medium. Alcohols seem to block cells in an early phase of starvation-induced differentiation, most likely by interfering with processes important for regulation of sigma(s) such as cell density signals and sensing the nutrient content of the medium.