Effects of polyhydroxy compounds on the structure and activity of alpha-chymotrypsin

The effects of glycerol, polyethylene glycol, fructose, glucose, sorbitol, and saccharose on the conformation and catalytic activity of alpha-chymotrypsin were studied in 0.1 M sodium phosphate buffer and buffered aqueous 60% ethanol (pH 8.0). The enzyme activity was practically completely lost within 10 min in 60% ethanol, but in the presence of stabilizers the activity was retained. With the exception of polyethylene glycol, the stabilizing effect decreased with increase of the incubation time. The preservation of the catalytic activity was accompanied by changes in the secondary and tertiary structures of alpha-chymotrypsin.     

Establishment of a semi-automated pathogen DNA isolation from whole blood and comparison with commercially available kits

Molecular methods for bacterial pathogen identification are gaining increased importance in routine clinical diagnostic laboratories. Achieving reliable results using DNA based technologies is strongly dependent on pre-analytical processes including isolation of target cells and their DNA of high quality and purity. In this study a fast and semi-automated method was established for bacterial DNA isolation from whole blood samples and compared to different commercially available kits: Looxster, MolYsis kit, SeptiFast DNA isolation method and standard EasyMAG protocol. The newly established, semi-automated method utilises the EasyMAG device combined with pre-processing steps comprising human cell lysis, centrifugation and bacterial pellet resuspension. Quality of DNA was assessed by a universal PCR targeting the 16S rRNA gene and subsequent microarray hybridisation. The DNA extractions were amplified using two different PCR-mastermixes, to allow comparison of a commercial mastermix with a guaranteed bacterial DNA free PCR mastermix. The modified semi-automated EasyMAG protocol and the Looxster kit gave the most sensitive results. After hybridisation a detection limit of 10¹ to 10² bacterial cells per mL whole blood was achieved depending on the isolation method and microbial species lysed. Human DNA present in the isolated DNA suspension did not interfere with PCR and did not lead to non-specific hybridisation events.     

Detection and identification of mycobacteria by mycolic acid analysis of sputum specimens and young cultures

Ziehl–Neelsen acid-fast staining and mycolic acid analysis of concentrated samples and Middlebrook 7H9 cultures were carried out on 127 sputum specimens to evaluate a rapid method for detecting and identifying mycobacteria by analyzing fluorescent derivatives of mycolic acids in concentrated sputum specimens and in Middlebrook 7H9 cultures and compare with mycobacterial detection using Lowenstein–Jensen (LJ) cultures. All samples were classified into five groups according to the number of acid-fast bacilli observed in the smear. The group of samples with 3+ acid-fast bacilli in the smear had the highest number of positive detections of mycolic acids in the concentrated samples and the Middlebrook 7H9 cultures (81.8 and 100%, respectively). The overall percentages of mycolic acid detection for both sample types were 43.2 and 91.3%, respectively. The mycolic acid analysis of the Middlebrook 7H9 cultures had the fewest false negative detections with respect to the LJ cultures. The analysis of fluorescent derivatives of mycolic acids, using HPLC, is useful for concentrated sputum samples with large number of bacilli (3+) and is preferred for Middlebrook 7H9 cultures, even for clinical specimens with a low number of bacilli. Furthermore, with this analytical method, the simultaneous detection and identification of mycobacteria is usually possible.     

Identification of phenolic compounds in isolated vacuoles of the medicinal plant Catharanthus roseus and their interaction with vacuolar class III peroxidase: an H₂O₂ affair?

Class III peroxidases (Prxs) are plant enzymes capable of using H(2)O(2) to oxidize a range of plant secondary metabolites, notably phenolic compounds. These enzymes are localized in the cell wall or in the vacuole, which is a target for secondary metabolite accumulation, but very little is known about the function of vacuolar Prxs. Here, the physiological role of the main leaf vacuolar Prx of the medicinal plant Catharanthus roseus, CrPrx1, was further investigated namely by studying its capacity to oxidize co-localized phenolic substrates at the expense of H(2)O(2). LC-PAD-MS analysis of the phenols from isolated leaf vacuoles detected the presence of three caffeoylquinic acids and four flavonoids in this organelle. These phenols or similar compounds were shown to be good CrPrx1 substrates, and the CrPrx1-mediated oxidation of 5-O-caffeoylquinic acid was shown to form a co-operative regenerating cycle with ascorbic acid. Interestingly, more than 90% of total leaf Prx activity was localized in the vacuoles, associated to discrete spots of the tonoplast. Prx activity inside the vacuoles was estimated to be 1809 nkat ml(-1), which, together with the determined concentrations for the putative vacuolar phenolic substrates, indicate a very high H(2)O(2) scavenging capacity, up to 9 mM s(-1). Accordingly, high light conditions, known to increase H(2)O(2) production, induced both phenols and Prx levels. Therefore, it is proposed that the vacuolar couple Prx/secondary metabolites represent an important sink/buffer of H(2)O(2) in green plant cells.     

miR-126 inhibits proliferation of small cell lung cancer cells by targeting SLC7A5

Despite intensive efforts to improve therapies, small cell lung cancer (SCLC) still has a dismal median survival of 18 months. Since miR-126 is under-expressed in the majority of SCLC tumors, we investigated the effect of miR-126 overexpression on the proliferation and cell cycle distribution of H69 cells. Our results demonstrate that miR-126 inhibits proliferation of H69 cells, by delaying the cells in the G1 phase. Short interfering RNA (siRNA) mediated suppression of SLC7A5, a predicted target of mir-126, has the same effect on H69 cells. We also show for the first time that SLC7A5 is a direct target of miR-126.     

Essential oil of Lepechinia schiedeana (Lamiaceae) from Costa Rica

The composition of the essential oil isolated by steam distillation from aerial parts of the Costa Rican herb Lepechinia schiedeana (Schlecht) Vatke (Lamiaceae) collected in El Empalme, Costa Rica, was determined by capillary gas chromatography (GC) and coupled gas chromatography-mass spectrometry (GC-MS) analyses. Fifty-one components were identified corresponding ca. 93% of the oil. The major components were beta-pinene (26.6%), cis-pinocamphone (25.1%), delta-3-carene (6.1%), trans-pinocamphone (4.0%), camphor (3.8%) and beta-caryophyllene (3.7%).     

Effects of pituitary adenylate cyclase-activating polypeptide on the cyclooxygenase pathway of rat platelets and on platelet aggregation

Several data suggest that pituitary adenylate cyclase-activating polypeptide (PACAP) is involved in the regulation of local circulation. One possible role of PACAP in the regulation of circulation is that, it may modify the cyclooxygenase pathway of the arachidonate cascade in platelets. Our study was designed to study the effect of PACAP on the cyclooxygenase pathway of rat platelets and on platelet aggregation. PACAP (10(-7) and 10(-6) M) significantly inhibited the cyclooxygenase pathway of platelets, mostly the thromboxane synthesis. Pretreatment with a PACAP receptor antagonist, PACAP(6-38), or with an inhibitor of protein kinase A, H-89, shows that the effects of PACAP on the cyclooxygenase pathway were diminished. In the aggregation studies, PACAP inhibited both the arachidonic acid-induced and the thrombin-induced platelet aggregation. It can be concluded that PACAP inhibits the cyclooxygenase pathway of rat platelets via a specific PACAP receptor-activated, cAMP-dependent pathway, and these effects of PACAP are involved in the inhibition of platelet aggregation.     

Foxb1-driven Cre expression in somites and the neuroepithelium of diencephalon, brainstem, and spinal cord

We have knocked-in Cre-IRES-EGFP in the Foxb1 locus by homologous recombination in embryonic stem cells. We removed the PGK-neo cassette (which was flanked by FRT sequences) by crossing with the FLPeR deleter mouse. The Foxb1(Cre) line showed Cre recombinase activity as well as EGFP fluorescence reproducing Foxb1 expression accurately. By crossing Foxb1(Cre) mice with the ROSA26R and Z/AP mouse reporter lines we have been able to trace the lineage of Foxb1-expressing cells. Early transient expression of Foxb1 in the paraxial mesoderm translates into labeling of the somites. In the central nervous system (CNS), the Foxb1 lineage includes the thalamus and mammillary body (hypothalamus), brainstem, and the ventral spinal cord and floor plate.     

Cell surface antigens reacting with antiretrovirus sera on normal mouse spleen cells: a flow cytometric study

The development of erythroleukemia in Balb/c mice injected with Rauscher leukemia virus has been followed by indirect immunofluorescence technique and flow cytometry, using antiserum against disrupted of virions. The progression of the disease was accompanied by a great increase in the number of large, immunofluorescence positive cells. A subpopulation of normal spleen cells was also highly positive. The expression of the antigens in normal cells was examined in relation to the cell cycle. The majority of the S-G2/M phase cells were found in the antigen positive compartment of larger cells. A two-color analysis of immunofluorescence and DNA content revealed that the distribution of antigen expression in G1 phase was broad, gradually decreasing from a low-intensity mode. The cell with double DNA content distributed evenly around a modus of five-fold higher intensity. In experiments with stimulated bone-marrow cells, superiority of S-phase cells in anti-Rauscher serum binding was found. Cell-surface gp70 antigen is suggested to be involved in this cell-cycle dependent binding of antibodies by normal cells.