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.     

Quantification of total mitochondrial DNA and the 4977-bp common deletion in Pearson's syndrome lymphoblasts using a fluorogenic 5'-nuclease (TaqMan) real-time polymerase chain reaction assay and plasmid external calibration standards

This study describes a multiplex real-time polymerase chain reaction (PCR) assay that quantifies total mitochondrial DNA (mtDNA(total)) and mtDNA bearing the 4977-base pair 'common deletion' (deltamtDNA4977) in lymphoblasts derived from an individual diagnosed with Pearson's syndrome. The method is unique in its use of plasmids as external quantification standards and its use of multiplex conditions. Standards are validated by comparison with purified mtDNA amplification curves and by the fact that curves are largely unaffected by nuclear DNA (nucDNA). Finally, slopes of standard curves and unknowns are shown to be similar to each other and to theoretical predictions. From these data, mtDNA(total) in these cells is calculated to be 3258 (+723/-592) copies per cell while deltamtDNA4977 averages 232 (+136/-86) copies per cell or 7% (+4.65/-2.81).     

Characterization of thymic progenitors in adult mouse bone marrow

Thymic cellularity is maintained throughout life by progenitor cells originating in the bone marrow. In this study, we describe adult mouse bone cells that exhibit several features characteristic of prothymocytes. These include 1) rapid thymic engraftment kinetics following i.v. transplantation, 2) dramatic expansion of thymic progeny, and 3) limited production of hemopoietic progeny other than thymocytes. The adult mouse bone marrow population that is depleted of cells expressing any of a panel of lineage-specific Ags, stem cell Ag-1 positive, and not expressing the Thy1.1 Ag (Thy1.1(-)) (Thy1.1(-) progenitors) can repopulate the thymus 9 days more rapidly than can hemopoietic stem cells, a rate of thymic repopulation approaching that observed with transplanted thymocytes. Additionally, Thy1.1(-) progenitors expand prolifically to generate thymocyte progeny comparable in absolute numbers to those observed from parallel hemopoietic stem cell transplants, and provide a source of progenitors that spans multiple waves of thymic seeding. Nevertheless, the Thy1.1(-) population yields relatively few B cells and rare myeloid progeny posttransplant. These observations describe the phenotype of an adult mouse bone marrow population highly enriched for rapidly engrafting, long-term thymocyte progenitors. Furthermore, they note disparity in B and T cell expansion from this lymphoid progenitor population and suggest that it contains the progenitor primarily responsible for seeding the thymus throughout life.     

NMR Studies of Pi-Containing Extracellular and Cytoplasmic Compartments in Brain

Abstract: The inorganic phosphate (P_i) NMR peak in brain has an irregular shape, which suggests that it represents more than a single homogeneous pool of P_i. To test the ability of the Marquardt-Levenberg (M-L) nonlinear curve fit algorithm software (Peak-Fit) to separate multiple peaks, locate peak centers, and estimate peak heights, we studied simulated P_i spectra with defined peak centers, areas, and signal-to-noise (S/N) ratios ranging from ∞ to 5.8. As the S/N ratio decreased below 15, the M-L algorithm located peak centers accurately when they were detected; however, small peaks tended to grow smaller and disappear, whereas the amplitudes of larger peaks increased. We developed an in vitro three-compartment model containing a mixture of P_i buffer, phosphocreatine, phosphate diester, and phosphate monoester (PME), portions of which were adjusted to three different pHs before addition of agar. Weighed samples of each buffered gel together with phospholipid extract and bone chips were placed in an NMR tube and covered with mineral oil. Following baseline correction, it was possible to separate the P_i peaks arising from the three compartments with different pH values if each peak made up 10–35% of total P_i area. In vivo, we identified the plasma compartment by intraarterial infusion of P_i. It was assumed that intracellular compartments contained high-energy phosphates and took up glucose. Based on these assumptions we subjected the brains to complete ischemia and observed that P_i compartments at pH 6.82, 6.92, 7.03, and 7.13 increased markedly in amplitude. If the brain cells took up and phosphorylated 2-deoxyglucose (2-DG), 2-DG-6-phosphate (2-DG-6-P) would appear in the PME portion of the spectrum ionized according to pH_i. Four 2-DG-6-P peaks with calculated pH values of 6.86, 6.94, 7.04, and 7.15 did appear in the spectrum, thereby confirming that the four larger P_i peaks represented intracellular spaces.     

Burkholderia pseudomallei γ-carbonic anhydrase is strongly activated by amino acids and amines

Activation of the γ-class carbonic anhydrase (CAs, EC 4.2.1.1) from the pathogenic bacterium Burkholderia pseudomallei (BpsγCA) with a series of natural and non-natural amino acids and aromatic/heterocyclic amines has been investigated. The best BpsγCA activators were d-His, l-DOPA, d-Trp, 4-amino-l-Phe, dopamine, 2-(2-aminoethyl)pyridine, 2-aminoethyl-piparazine/morpholine and l-adrenaline, which showed activation constants ranging between 9 and 86 nM. The least effective activators were l-His, l-Phe and 2-pyridyl-methylamine, with K_As in the range of 1.73–24.7 μM. As little is known about the role of γ-CAs in the lifecycle and virulence of this saprophytic bacterium, this study may shed some light on such phenomena. This is the first CA activation study of a γ-CA from a pathogenic bacterium, the only other such study being on the enzyme discovered in the archaeon Methanosarcina thermophila, Cam.     

Complex Biventricular Pacing - A Case Series

It is established that cardiac resynchronisation therapy (CRT) reduces mortality and hospitalisation and improves functional class in patients with NYHA class 3-4 heart failure, an ejection fraction of ≤ 35% and a QRS duration of ≥ 120ms. Recent updates in the American guidelines have expanded the demographic of patients in whom CRT may be appropriate. Here we present two cases of complex CRT; one with a conventional indication but occluded central veins and the second with a novel indication for CRT post cardiac transplant.     

Getting more from less: algorithms for rapid protein identification with multiple short peptide sequences

We describe two novel sequence similarity search algorithms, FASTS and FASTF, that use multiple short peptide sequences to identify homologous sequences in protein or DNA databases. FASTS searches with peptide sequences of unknown order, as obtained by mass spectrometry-based sequencing, evaluating all possible arrangements of the peptides. FASTF searches with mixed peptide sequences, as generated by Edman sequencing of unseparated mixtures of peptides. FASTF deconvolutes the mixture, using a greedy heuristic that allows rapid identification of high scoring alignments while reducing the total number of explored alternatives. Both algorithms use the heuristic FASTA comparison strategy to accelerate the search but use alignment probability, rather than similarity score, as the criterion for alignment optimality. Statistical estimates are calculated using an empirical correction to a theoretical probability. These calculated estimates were accurate within a factor of 10 for FASTS and 1000 for FASTF on our test dataset. FASTS requires only 15-20 total residues in three or four peptides to robustly identify homologues sharing 50% or greater protein sequence identity. FASTF requires about 25% more sequence data than FASTS for equivalent sensitivity, but additional sequence data are usually available from mixed Edman experiments. Thus, both algorithms can identify homologues that diverged 100 to 500 million years ago, allowing proteomic identification from organisms whose genomes have not been sequenced.     

Matrix metalloproteinase expression and activity following prostaglandin F(2 alpha)-induced luteolysis

Luteal tissue contains matrix metalloproteinases (MMPs) that cleave specific components of the extracellular matrix (ECM) and are inhibited by tissue inhibitors of metalloproteinases (TIMPs). We previously reported a decrease in luteal TIMP-1 within 15 min of prostaglandin F(2 alpha) (PGF(2 alpha))-induced luteolysis. An increase in the MMP:TIMP ratio may promote ECM degradation and apoptosis, as observed in other tissues that undergo involution. The objectives of these experiments were to determine whether 1) PGF(2 alpha) affects expression of mRNA encoding fibrillar collagenases (MMP-1 and -13), gelatinases A and B (MMP-2 and -9), membrane type (mt)-1 MMP (MMP-14), stromelysin (MMP-3), and matrilysin (MMP-7), and 2) PGF(2 alpha) increases MMP activity during PGF(2 alpha)-induced luteolysis in sheep. Corpora lutea (n = 3-10/time point) were collected at 0, 15, and 30 min and 1, 2, 4, 6, 12, 24, and 48 h after PGF(2 alpha) administration. Northern blot analysis confirmed the presence of all MMPs except MMP-9. Expression of mRNA for the above MMPs (except MMP-2) increased significantly (P < 0.05) by 30 min, and all MMPs increased significantly (P < 0.05) by 6 h after PGF(2 alpha) administration. Expression of MMP-14 mRNA increased significantly (P < 0.05) by 15 min post-PGF(2 alpha) and remained elevated through 48 h. MMP activity in luteal homogenates (following proenzyme activation and inactivation of inhibitors) was increased significantly (P < 0.05) by 15 min and remained elevated through 48 h post-PGF(2 alpha). MMP activity was localized (in situ zymography) to the pericellular area of various cell types in the 0-h group and was markedly increased by 30 min post-PGF(2 alpha). MMP mRNA expression and activity were significantly increased following PGF(2 alpha) treatment. Increased MMP activity may promote ECM degradation during luteolysis.     

Human NK cell-mediated cytotoxicity triggered by CD86 and Gal alpha 1,3-Gal is inhibited in genetically modified porcine cells

Delayed xenograft rejection is a major hurdle that needs to be addressed to prolong graft survival in pig-to-primate xenotransplantation. NK cell activation has been implicated in delayed xenograft rejection. Both Ab-dependent and independent mechanisms are responsible for the high susceptibility of porcine cells to human NK cell-mediated cytotoxicity. Previous reports demonstrated a role of Galalpha1,3-Gal Ag in triggering the Ab-independent responses. We hypothesize that expression of CD80 and/or CD86 on porcine cells may also play a role in NK cell activation as human NK cells express a variant of CD28. Our initial analysis showed that porcine endothelial cells and fibroblasts express CD86, but not CD80. Genetic engineering of these cells to express hCD152-hCD59, a chimeric molecule designed to block CD86 in cis, was accompanied by a reduction in susceptibility to human NK cell-mediated cytotoxicity. The use of a specific anti-porcine CD86-blocking Ab and the NK92 and YTS cell lines further confirmed the involvement of CD86 in triggering NK cell-mediated lysis of porcine cells. Maximal protection was achieved when hCD152-hCD59 was expressed in H transferase-transgenic cells, which show reduced Galalpha1,3-Gal expression. In this work, we describe two mechanisms of human NK cell-mediated rejection of porcine cells and demonstrate that genetically modified cells resist Ab-independent NK cell-mediated cytotoxicity.