Administration of a nitric oxide synthase inhibitor counteracts prostaglandin F2-induced luteolysis in cattle

The objective of this study was to determine whether nitric oxide (NO) is produced locally in the bovine corpus luteum (CL) and whether NO mediates prostaglandin F2alpha (PGF2alpha)-induced regression of the bovine CL in vivo. The local production of NO was determined in early I, early II, mid, late, and regressed stages of CL by determining NADPH-d activity and the presence of inducible and endothelial NO synthase immunolabeling. To determine whether inhibition of NO production counteracts the PGF2alpha-induced regression of the CL, saline (10 ml/h; n = 10) or a nonselective NOS inhibitor (Nomega-nitro-l-arginine methyl ester dihydrochloride [L-NAME]; 400 mg/h; n = 9) was infused for 2 h on Day 15 of the estrous cycle into the aorta abdominalis of Holstein/Polish Black and White heifers. After 30 min of infusion, saline or cloprostenol, an analogue of PGF2alpha (aPGF2alpha; 100 microg) was injected into the aorta abdominalis of animals infused with saline or L-NAME. NADPH-diaphorase activity was present in bovine CL, with the highest activity at mid and late luteal stages (P < 0.05). Inducible and endothelial NO synthases were observed with the strongest immunolabeling in the late CL (P < 0.05). Injection of aPGF2alpha increased nitrite/nitrate concentrations (P < 0.01) and inhibited P4 secretion (P < 0.05) in heifers that were infused with saline. Infusion of L-NAME stimulated P4 secretion (P < 0.05) and concomitantly inhibited plasma concentrations of nitrite/nitrate (P < 0.05). Concentrations of P4 in heifers infused with L-NAME and injected with aPGF2alpha were higher (P < 0.05) than in animals injected only with aPGF2alpha. The PGF2alpha analogue shortened the cycle length compared with that of saline (17.5 +/- 0.22 days vs. 21.5 +/- 0.65 days P < 0.05). L-NAME blocked the luteolytic action of the aPGF2alpha (22.6 +/- 1.07 days vs. 17.5 +/- 0.22 days, P < 0.05). These results suggest that NO is produced in the bovine CL. NO inhibits luteal steroidogenesis and it may be one of the components of an autocrine/paracrine luteolytic cascade induced by PGF2alpha.     

Identification and characterization of transposable elements of Paracoccus pantotrophus

We studied diversity and distribution of transposable elements residing in different strains (DSM 11072, DSM 11073, DSM 65, and LMD 82.5) of a soil bacterium Paracoccus pantotrophus (alpha-Proteobacteria). With application of a shuttle entrapment vector pMEC1, several novel insertion sequences (ISs) and transposons (Tns) have been identified. They were sequenced and subjected to detailed comparative analysis, which allowed their characterization (i.e., identification of transposase genes, terminal inverted repeats, as well as target sequences) and classification into the appropriate IS or Tn families. The frequency of transposition of these elements varied and ranged from 10(-6) to 10(-3) depending on the strain. The copy number, localization (plasmid or chromosome), and distribution of these elements in the Paracoccus species P. pantotrophus, P. denitrificans, P. methylutens, P. solventivorans, and P. versutus were analyzed. This allowed us to distinguish elements that are common in paracocci (ISPpa2, ISPpa3--both of the IS5 family--and ISPpa5 of IS66 family) as well as strain-specific ones (ISPpa1 of the IS256 family, ISPpa4 of the IS5 family, and Tn3434 and Tn5393 of the Tn3 family), acquired by lateral transfer events. These elements will be of a great value in the design of new genetic tools for paracocci, since only one element (IS1248 of P. denitrificans) has been described so far in this genus.     

Immunological characterization of the Campylobacter jejuni 72Dz/92 cjaD gene product and its fusion with B subunit of E. coli LT toxin

Campylobacter jejuni 72Dz/92 cjaD gene, orthologue of C. jejuni NCTC 11168 cj0113, C. jejuni M275 omp18 and C. jejuni ATCC 29428 omp18, has been cloned, sequenced and analysed from the viewpoint of its immunological attributes. Neither the 5' nor 3' fragment of the cjaD encodes protein capable of reacting with anti-Campylobacter antibodies. Several fusions of the cjaD with eltB, which encodes B subunit of the E. coli LT toxin, have been constructed. The hybrid proteins, which differ in respect to their cellular localization, retain the ability to react with GM1 and are recognized by the antibodies specific for both moieties of the proteins. The fusion protein equipped with signal sequence, reveals a stronger affinity to GM1 than its equivalent which is unable to cross the inner membrane. Two recombinant plasmids (pUWM405 expressing both LTB and CjaD proteins and pUWM299 containing cjaD gene fused into 3' end of Escherichia coli eltB gene lacking signal sequence) were introduced into avirulent Salmonella enterica serovar Typhimurium strain where they are stably maintained.     

Biological properties of a new fluorescent biphalin fragment analogue

Previous studies of structure-activity of biphalin defined fragments which expressed the full biological potency of the parent compound. The most simple fragment was Tyr-D-Ala-Gly-Phe-NH-NH<--X, where X=Phe, but it also could be other hydrophobic amino acids. This paper presents data that replacement of the phenylalanine with a dansyl (X=DNS) groups gives an analogue (AA2016) that fully preserves the high affinity of the initial analogue for both mu and delta opioid receptors. In the tail flick test in rats, intrathecal injection of the compound produces strong antinociception, comparable to the parent biphalin. Because AA2016 contains a strong fluorescent group, it can be a very useful tool for prospective studies in vivo, including biological barrier permeability, tissue distribution, metabolism and receptor-ligand complex formation.     

Cytological and morphological differences between two species of the genus Tettigonia (Orthoptera,Tettigoniidae) from Korea

Tettigonia ussuriana and T. dolichopoda maritima differ in the length of tegmina, details in venation, and in females in details of the subgenital plate. The two species of the genus Tettigonia have the same number and morphology of autosomes but a different morphology of the X chromosome: in T. ussuriana it is metacentric, whereas in T. dolichopoda maritima acrocentric. In both species, euchromatic zones and breaks of one or to chromatids during meiosis and mitosis in the X chromosome were observed. Additionally, B chromosomes were noted in most individuals of both species.     

Optimization of solid-phase microextraction conditions for gas chromatographic determination of ethanol and other volatile compounds in blood

A procedure for the determination of acetaldehyde, acetone, methanol, ethanol, 1-propanol and 2-propanol in blood was developed. Separation of analytes was carried out on DB-wax capillary column (l = 30 m, I.D. = 0.32 mm, dF = 0.5 microm) at 40 degrees C, hydrogen was used as a carrier gas (at 30 kPa) and FID as a detector. Quantification was performed with the use of 2-butanol as an internal standard. Headspace solid-phase microextraction was applied as the sample preparation technique. The usefulness of most commercially available fiber coatings was checked and 65 microm Carbowax/DVB proved most effective. Microextraction was carried out from the headspace at 60 degrees C for 10 min. The sample was stirred at 750 rpm. In order to improve the extraction efficiency of analytes, salting-out agents were also applied. Potassium carbonate turned out to be the most efficient. A 1.0-g amount of this salt and 0.1 ml of I.S. were added to 0.5 ml of sample. Validation of the worked-out method was performed. For each analyte, the limits of detection and quantification, linearity, working range, accuracy and precision were determined or tested.     

Influence of nitric oxide on the secretory function of the bovine corpus luteum: dependence on cell composition and cell-to-cell communication

The objective of the present study was to investigate the role of cell-to-cell contact in the influence of nitric oxide (NO) on the secretory function of the bovine corpus luteum (CL). In Experiment 1, separate small luteal cells (SLC) or large (LLC) luteal cells were perfused with 100 micro M spermineNONOate, a NO donor, or with 100 micro M Nomega-nitro-L-arginine methyl ester (L-NAME), a NO synthase (NOS) inhibitor; in Experiment 2, a mixture of LLC and SLC and endothelial cells was cultured and incubated with spermineNONOate or L-NAME; in Experiment 3, spermineNONOate was perfused into the CL (100 mg/4 hr) by a microdialysis system in vivo. Perfusion of isolated SLC and LLC with the NO donor or NOS inhibitor (Experiment 1) did not affect (P > 0.05) secretion of progesterone (P(4)) or oxytocin (OT). L-NAME perfusion increased (P < 0.05) leukotriene C(4) (LTC(4)) secretion by both SLC and LLC cells. Treatment of mixtures of luteal cells with an NO donor (Experiment 2) significantly decreased (P < 0.001) secretion of P(4) and OT and increased (P < 0.001) production of prostaglandin F(2alpha) (PGF(2alpha)) and LTC(4). L-NAME stimulated (P < 0.001) P(4) secretion, but did not influence (P > 0.05) OT, PGF(2alpha) or LTC(4) production. Intraluteal administration (Experiment 3) of spermineNONOate increased (P < 0.001) LTC(4) and PGF(2alpha), decreased OT, but did not change P(4) levels in perfusate samples. These data indicate that cell-to-cell contact and cell composition play important roles in the response of bovine CL to treatment with NO donors or NOS inhibitors, and that paracrine mechanisms are required for the full secretory response of the CL in NO action. Endothelial cells appear to be required for the full secretory response of the CL to NO.     

Crystal structure of the class D beta-lactamase OXA-10

We report the crystal structure of a class D beta-lactamase, the broad spectrum enzyme OXA-10 from Pseudomonas aeruginosa at 2.0 A resolution. There are significant differences between the overall fold observed in this structure and those of the evolutionarily related class A and class C beta-lactamases. Furthermore, the structure suggests the unique, cation mediated formation of a homodimer. Kinetic and hydrodynamic data shows that the dimer is a relevant species in solution and is the more active form of the enzyme. Comparison of the molecular details of the active sites of the class A and class C enzymes with the OXA-10 structure reveals that there is no counterpart in OXA-10 to the residues proposed to act as general bases in either of these enzymes (Glu 166 and Tyr 150, respectively). Our structures of the native and chloride inhibited forms of OXA-10 suggest that the class D enzymes have evolved a distinct catalytic mechanism for beta-lactam hydrolysis. Clinical variants of OXA-10 are also discussed in light of the structure.