Substrate and metal specificity in the enzymic synthesis of cyclic monoterpenes from geranyl and neryl pyrophosphate

A partially purified enzyme (carbocyclase) from the flavedo of Citrus limonum formed α-pinene, β-pinene, limonene, and γ-terpinene from geranyl pyrophosphate (GPP) and neryl pyrophosphate. The maximum specific activities obtained were 7.0 and 3.6 nmol/ min/mg, respectively. Cross-inhibition by the two substrates were observed and the ability to utilize neryl pyrophosphate was almost completely lost with aging. Citronellyl pyrophosphate and dimethylallyl pyrophosphate were the most effective inhibitors of carbocyclase. Isopentenyl pyrophosphate, the monophosphate esters of nerol and geraniol, as well as inorganic pyrophosphate were much less effective inhibitors. The enzyme had an absolute requirement for Mn²⁺. It could be replaced with about 2% effectiveness by Mg²⁺ and Co²⁺. Kinetic studies showed that the observed reaction rate correlates with the calculated concentration of the GPP (Mn²⁺)₂ species. Previous evidence with nonenzymatic reactions and the results presented support the view that the mechanism of carbocyclase may be the intramolecular analog of prenyltransferase.     

Update of AMmtDB: a database of multi-aligned Metazoa mitochondrial DNA sequences

The AMmtDB database (http://bighost.area.ba.cnr.it/mitochondriome) has been updated by collecting the multi-aligned sequences of Chordata and Invertebrata mitochondrial genes coding for proteins and tRNAs. Links to the multi-aligned mtDNA intraspecies variants, collected in VarMmtDB at the Mitochondriome web site, have been introduced. The genes coding for proteins are multi-aligned based on the translated sequences and both the nucleotide and amino acid multi-alignments are provided. AMmtDB data selected through SRS can be viewed and managed using GeneDoc or other programs for the management of multi-aligned data depending on the user’s operative system. The multiple alignments have been produced with CLUSTALW and PILEUP programs and then carefully optimized manually.     

Impact of Human Cytomegalovirus Infection and its Immune Response on Survival of Patients with Ovarian Cancer

Human cytomegalovirus (HCMV) has been detected in various types of tumors. We studied the prevalence of HCMV in ovarian cancer and its relation to clinical outcome. Paraffin-embedded tissues obtained prospectively from 45 patients with ovarian cancer and 30 patients with benign ovarian cystadenoma were analyzed for expression of HCMV immediate-early protein (IE) and HCMV tegument protein (pp65) by immunohistochemistry. Plasma was analyzed for HCMV serology. HCMV-IgG levels were higher in patients with ovarian cancer or benign cystadenoma than in age-matched controls (P?=?.002, P?<?.0001, respectively). HCMV IgM was detected in 12% of ovarian cancer patients and 3% of patients with benign tumors but was absent in controls. In patients with ovarian cancer, higher IgG levels were associated with better outcomes (P?=?.04). Extensive HCMV-IE protein expression was detected in 75% of ovarian cancers and 26% of benign tumors; pp65 was detected in 67% of ovarian cancers and 14% of benign tumors. A higher grade of HCMV infection was associated with higher stage of disease. Extensive HCMV-pp65 expression was associated with shorter median overall survival than focal expression (39 versus 42.5?months, P?=?.03). At study closure, 58% of ovarian cancer patients with focal pp65 expression were alive versus 27% of patients with extensive pp65 expression (P?=?.03). Thus, HCMV proteins are detected at different levels in ovarian tumors and benign cystadenomas. Ovarian cancer patients with focal HCMV-pp65 expression in their tumors and high IgG levels against HCMV lived longer, highlighting a need for in-depth studies of the oncomodulatory role of HCMV in ovarian cancer.     

Paddyfield Warbler,Acrocephalus agricola,at Van Gölü, eastern Turkey

Abstract

A Paddyfield Warbler was mist-netted at Van in eastern Anatolia on 8.5 1987. This is the second record for Turkey.     

A Comparative Study of Protocols for Mouse Embryonic Stem Cell Culturing

Most stem cell laboratories still rely on old culture methods to support the expansion and maintenance of mouse embryonic stem (ES) cells. These involve growing cells on mouse embryonic fibroblast feeder cells or on gelatin in media supplemented with fetal bovine serum and leukemia inhibitory factor (LIF). However, these techniques have several drawbacks including the need for feeder-cells and/or use of undefined media containing animal derived components. Culture of stem cells under undefined conditions can induce spontaneous differentiation and reduce reproducibility of experiments. In recent years several new ES cell culture protocols, using more well-defined conditions, have been published and we have compared the standard culture protocols with two of the newly described ones: 1) growing cells in semi-adherence in a medium containing two small molecule inhibitors (CHIR99021, PD0325901) and; 2) growing cells in a spheroid suspension culture in a defined medium containing LIF and bFGF. Two feeder-dependent mouse ES (mES) cell lines and two cell lines adapted to feeder-independent growth were used in the study. The overall aim has not only been to compare self-renewal and differentiation capacity, but also ease-of-use and cost efficiency. We show that mES cells when grown adherently proliferate much faster than when grown in suspension as free-floating spheres, independent of media used. Although all the tested culture protocols could maintain sustained pluripotency after prolonged culturing, our data confirm previous reports showing that the media containing two chemical inhibitors generate more pure stem cell cultures with negligible signs of spontaneous differentiation as compared to standard mES media. Furthermore, we show that this medium effectively rescues and cleans up cultures that have started to deteriorate, as well as allow for effective adaption of feeder-dependent mES cell lines to be maintained in feeder-free conditions.     

Selective decrease in paracellular conductance of tight junctions: role of the first extracellular domain of claudin-5

Claudin-5 is a protein component of many endothelial tight junctions, including those at the blood-brain barrier, a barrier that limits molecular exchanges between the central nervous system and the circulatory system. To test the contribution of claudin-5 to this barrier function of tight junctions, we expressed murine claudin-5 in Madin-Darby canine kidney II cells. The result was a fivefold increase in transepithelial resistance in claudin-5 transductants and a reduction in conductance of monovalent cations. However, the paracellular flux of neither neutral nor charged monosaccharides was significantly changed in claudin-5 transductants compared to controls. Therefore, expression of claudin-5 selectively decreased the permeability to ions. Additionally, site-directed mutations of particular amino acid residues in the first extracellular domain of claudin-5 altered the properties of the tight junctions formed in response to claudin-5 expression. In particular, the conserved cysteines were crucial: mutation of either cysteine abolishted the ability of claudin-5 to increase transepithelial resistance, and mutation of Cys(64) strikingly increased the paracellular flux of monosaccharides. These new insights into the functions of claudin-5 at the molecular level in tight junctions may account for some aspects of the blood-brain barrier's selective permeability.