A Unique Pair of GATC Specific DNA Methyltransferases in <Emphasis Type="Italic">Mitsuokella multiacida</Emphasis>

Two GATC specific methylases together with Sau3AI isoschizomeric restriction endonuclease were partially characterized in Mitsuokella multiacida 46/5. This is the first report on the presence of solitary Dam methyltransferase alongside GATC specific restriction-modification system resulting in the unusual two-fold methylation of the GATC motifs.     

Phosphatidyl ethanolamine is essential for targeting the arginine transporter Can1p to the plasma membrane of yeast

In continuation of our previous study, we show that phosphatidyl ethanolamine (PE) depletion affects, in addition to amino acid transporters, activities of at least two other proton motive force (pmf)-driven transporters (Ura4p and Mal6p). For Can1p, we demonstrate that the lack of PE results in a failure of the permease targeting to plasma membrane. Despite the pleiotropic effect of PE depletion, a specific role of PE in secretion of a defined group of permeases can be distinguished. Pmf-driven transporters are more sensitive to the lack of PE than other plasma membrane proteins.     

The place and role of serologic methods in detecting Helicobacter pylori infection

The aim of the study was to determine the place and role of serologic methods in detecting Helicobacter pylori (H. pylori) infection, on the basis of estimated enzyme-linked immunosorbent assay (ELISA) and complement fixation test (CFT) sensitivity and specificity. A total of 549 patients were included in the study. ELISA and CFT as serologic methods were compared with invasive methods (rapid urease test--CLO test, culture, histology). The sensitivity of serologic methods was above 90%, and their specificity was around 80%. Study results confirmed the value, reliability and usefulness of serologic methods in the detection of H. pylori infection.     

Structural basis of interactions between human glutamate carboxypeptidase II and its substrate analogs

Human glutamate carboxypeptidase II (GCPII) is involved in neuronal signal transduction and intestinal folate absorption by means of the hydrolysis of its two natural substrates, N-acetyl-aspartyl-glutamate and folyl-poly-γ-glutamates, respectively. During the past years, tremendous efforts have been made toward the structural analysis of GCPII. Crystal structures of GCPII in complex with various ligands have provided insight into the binding of these ligands, particularly to the S1′ site of the enzyme. In this article, we have extended structural characterization of GCPII to its S1 site by using dipeptide-based inhibitors that interact with both S1 and S1′ sites of the enzyme. To this end, we have determined crystal structures of human GCPII in complex with phosphapeptide analogs of folyl-γ-glutamate, aspartyl-glutamate, and γ-glutamyl-glutamate, refined at 1.50, 1.60, and 1.67 Å resolution, respectively. The S1 pocket of GCPII could be accurately defined and analyzed for the first time, and the data indicate the importance of Asn519, Arg463, Arg534, and Arg536 for recognition of the penultimate (i.e., P1) substrate residues. Direct interactions between the positively charged guanidinium groups of Arg534 and Arg536 and a P1 moiety of a substrate/inhibitor provide mechanistic explanation of GCPII preference for acidic dipeptides. Additionally, observed conformational flexibility of the Arg463 and Arg536 side chains likely regulates GCPII affinity toward different inhibitors and modulates GCPII substrate specificity. The biochemical experiments assessing the hydrolysis of several GCPII substrate derivatives modified at the P1 position, also included in this report, further complement and extend conclusions derived from the structural analysis. The data described here form an a solid foundation for the structurally aided design of novel low-molecular-weight GCPII inhibitors and imaging agents.     

STIM1/ORAI1-mediated Ca2+ Influx Regulates Enolase-1 Exteriorization

Tumor cells use broad spectrum proteolytic activity of plasmin to invade tissue and form metastatic foci. Cell surface-associated enolase-1 (ENO-1) enhances plasmin formation and thus participates in the regulation of pericellular proteolysis. Although increased levels of cell surface bound ENO-1 have been described in different types of cancer, the molecular mechanism responsible for ENO-1 exteriorization remains elusive. In the present study, increased ENO-1 protein levels were found in ductal breast carcinoma and on the cell surface of highly metastatic breast cancer cell line MDA-MB-231. Elevated cell surface-associated ENO-1 expression correlated with augmented MDA-MB-231 cell migratory and invasive properties. Exposure of MDA-MB-231 cells to LPS potentiated translocation of ENO-1 to the cell surface and its release into the extracellular space in the form of exosomes. These effects were independent of de novo protein synthesis and did not require the classical endoplasmic reticulum/Golgi pathway. LPS-triggered ENO-1 exteriorization was suppressed by pretreatment of MDA-MB-231 cells with the Ca²⁺ chelator BAPTA or an inhibitor of endoplasmic reticulum Ca²⁺-ATPase pump, cyclopiazonic acid. In line with these observations, the stromal interaction molecule (STIM) 1 and the calcium release-activated calcium modulator (ORAI) 1-mediated store-operated Ca²⁺ entry were found to regulate LPS-induced ENO-1 exteriorization. Pharmacological blockage or knockdown of STIM1 or ORAI1 reduced ENO-1-dependent migration of MDA-MB-231 cells. Collectively, our results demonstrate the pivotal role of store-operated Ca²⁺ channel-mediated Ca²⁺ influx in the regulation of ENO-1 exteriorization and thus in the modulation of cancer cell migratory and invasive properties.     

Cytotoxic activity of proflavine diureas: synthesis, antitumor, evaluation and DNA binding properties of 1',1'-(acridin-3,6-diyl)-3',3'-dialkyldiureas

The synthesis of novel 1',1'-(acridin-3,6-diyl)-3',3'-dialkyldiureas was reported. Their biological activity to inhibit cell proliferation was assessed by a MTT assay on two cell lines, HeLa and HCT-116, at micromolar concentration. 1',1'-(Acridin-3,6-diyl)-3',3'-dihexyldiurea hydrochloride was active on a HCT-116 cell line with an IC(50) value of 3.1 microM. The interaction of these compounds with calf thymus DNA was investigated by a variety of spectroscopic techniques including UV-vis, fluorescence and CD spectroscopy. From spectrofluorimetric titrations, binding constants for the DNA-drug complexes were determined (K=0.9-4.2x10(5) M(-1)). Antiproliferative activity of synthesized derivatives might be related to their intercalation into DNA.     

Can pyrene probes be used to measure lateral pressure profiles of lipid membranes? Perspective through atomistic simulations

The lateral pressure profile of lipid bilayers has gained a lot of attention, since changes in the pressure profile have been suggested to shift the membrane protein conformational equilibrium. This relation has been mostly studied with theoretical methods, especially with molecular dynamics simulations, since established methods to measure the lateral pressure profile experimentally have not been available. The only experiments that have attempted to gauge the lateral pressure profile have been done by using di-pyrenyl-phosphatidylcholine (di-pyr-PC) probes. In these experiments, the excimer/monomer fluorescence ratio has been assumed to represent the lateral pressure in the location of the pyrene moieties. Here, we consider the validity of this assumption through atomistic molecular dynamics simulations in a DOPC (dioleoylphosphatidylcholine) membrane, which hosts di-pyr-PC probes with different acyl chain lengths. Based on the simulations, we calculate the pyrene dimerization rate and the lateral pressure at the location of the pyrenes. The dimerization rates are compared with the results of di-pyr-PC probes simulated in vacuum. The comparison indicates that the lateral pressure is not the dominant determinant of the excimer/monomer fluorescence ratio. Thus, the results do not support the usage of di-pyr-PC molecules to measure the shape of the lateral pressure profile. We yet discuss how the probes could potentially be exploited to gain qualitative insight of the changes in pressure profile when lipid composition is altered.     

Proteomics of the aqueous humor in healthy New Zealand rabbits

There are several physiological roles postulated for aqueous humor, a liquid located in the anterior and posterior chamber of the eye, such as maintenance of the intraocular pressure, provision of nutrients, and removal of metabolic waste from neighboring tissues and provision of an immune response and protection during inflammation and infection. To link these function to specific or classes of proteins, identification of the aqueous humor proteome is essential. Aqueous humor obtained from healthy New Zealand white rabbits was analyzed using three synergistic protein separation methods: 1-D gel electrophoresis, 2-DE, and 1-DLC (RPLC) prior to protein identification by MS. As each of these separation methods separates intact proteins based on different physical properties (pIs, molecular weights, hydrophobicity, solubility, etc.) the proteome coverage is expanded. This was confirmed, since overlap between all three separation technologies was only about 8.2% with many proteins found uniquely by a single method. Although the most dominant protein presented in normal aqueous humor is albumin, by using this extensive separation/MS strategy, additional proteins were identified in total amount of 98 nonredundant proteins (plus an additional ten proteins for consideration). This expands the current protein identifications by approximately 65%. The aqueous humor proteome comprises a specific selection of cellular and plasma based proteins and can almost exclusively be divided into four functional groups: cell-cell interactions/wound healing, proteases and protease inhibitors, antioxidant protection, and antibacterial/anti-inflammatory proteins.     

Effect of sodium humate on swelling and Germination of winter wheat

V práci byl studován vliv humátu sodného na bub?ení a klí?ení ozimé p? enice Py?elka (Triticum vulgare Vill.) a sledována změna intensity dýchání bub?ících obilek v prvých 24 hodinách bub?ení. Bylo zji?těno, ?e Na-humát v koncentraci 100 mg/ml urychluje v prvé fázi bub?oní p?íjem vody bub?ícími obilkami. Tím, ?e obilky p?ijmou d?ive dostate?né mno?ství vody, dochází u nich d?íve k aktivaci enzymatických systém?, zabezpe? ujících zdárný pr?běh klí?ení, co? se projeví zvý?ením intensity dý chání. Energie uvolněná dýcháním m??e být vyu?ita k rychlej?ímu r?stu embrya, co? se projeví morfologicky v rychlosti klí?ení.