Progesterone metabolism in bovine endometrial cells and the effect of metabolites on the responsiveness of the cells to OT-stimulation of PGF2alpha

Oxytocin receptor (OTR) expression is suppressed by progesterone (P4) during the luteal phase of the estrous cycle and then it increases at the time of luteolysis, but its regulation is still not completely understood. The objective of this work was to characterize P4 metabolism by endometrial cells in vitro and determine if metabolites were able to modify prostaglandin secretion in response to oxytocin (OT). Endometrial epithelial and stromal cells were incubated with 3H-P4 or 3H-pregnenolone (P5) for 6 or 24 h. Metabolites in the medium were separated by HPLC. The results showed that P4 and P5 were converted to two major polar metabolites and a less polar metabolite that was identified as 5alpha- or 5beta-pregnanedione by LC/MS. Progesterone metabolism was similar in both stromal and epithelial cells. To determine if 5alpha- or 5beta-pregnanedione were able to modify PGF(2)alpha synthesis, cells were cultured with P4, 5alpha- or 5beta-pregnanedione (100 ng ml(-1)) for 48 h and then each group of cells was incubated for a further 4-6 h with or without OT (200 ng ml(-1)). Results showed that only P4 caused significant (P<0.001) increase in basal, but not OT-stimulated, PGF(2)alpha synthesis. OT binding assays showed no significant effect of progesterone or its metabolites on OTR concentration. In conclusion, bovine endometrial cells are able to metabolize pregnenolone and progesterone but neither 5alpha- nor 5beta-pregnanedione altered prostaglandin synthesis or OTR number in endometrial epithelial cells. These data suggest that 5-pregnanediones do not play a role in the regulation OT-stimulated PGF(2)alpha secretion during the bovine estrous cycle.     

A comparative evaluation of EPR and OxyLite oximetry using a random sampling of pO(2) in a murine tumor

Methods currently available for the measurement of oxygen concentrations (oximetry) in viable tissues differ widely from each other in their methodological basis and applicability. The goal of this study was to compare two novel methods, particulate-based electron paramagnetic resonance (EPR) and OxyLite oximetry, in an experimental tumor model. EPR oximetry uses implantable paramagnetic particulates, whereas OxyLite uses fluorescent probes affixed on a fiber-optic cable. C3H mice were transplanted with radiation-induced fibrosarcoma (RIF-1) tumors in their hind limbs. Lithium phthalocyanine (LiPc) microcrystals were used as EPR probes. The pO(2) measurements were taken from random locations at a depth of approximately 3 mm within the tumor either immediately or 48 h after implantation of LiPc. Both methods revealed significant hypoxia in the tumor. However, there were striking differences between the EPR and OxyLite readings. The differences were attributed to the volume of tissue under examination and the effect of needle invasion at the site of measurement. This study recognizes the unique benefits of EPR oximetry in terms of robustness, repeatability and minimal invasiveness.     

Akt activation and augmented fibronectin production in hyperhexosemia

Dysmetabolic state in diabetes may lead to augmented synthesis of extracellular matrix (ECM) proteins. In the endothelial cells, we have previously demonstrated that glucose-induced fibronectin (FN) production and that of its splice variant, EDB(+)FN, is regulated by protein kinase B (PKB, also known as Akt). In this study, we investigated the role of Akt1 in ECM protein production in the organs affected by chronic diabetic complications. We studied Akt1/PKBalpha knockout mice and wild-type control littermates. To avoid confounding effects of systemic insulin, we used 30% galactose feeding to induce hyperhexosemia for 8 wk starting at 6 wk of age. We investigated FN mRNA, EDB(+)FN mRNA, and transforming growth factor (TGF)-beta mRNA expression, Akt phosphorylation, Akt kinase activity, and NF-kappaB and AP-1 activation in the retina, heart, and kidney. Renal and cardiac tissues were histologically examined. Galactose feeding caused significant upregulation of FN, EDB(+)FN, and TGF-beta in all tissues. FN protein levels paralleled mRNA. Such upregulation were prevented in Akt1-deficient galactose-fed mice. Galactose feeding caused ECM protein deposition in the glomeruli and in the myocardium, which was prevented in the Akt knockout mice. NF-kappaB and AP-1 activation was pronounced in galactose-fed wild-type mice and prevented in the galactose-fed Akt1/PKBalpha-deficient group. In the retina and kidney, Ser473 was the predominant site for Akt phosphorylation, whereas in the heart it was Thr308. Parallel experiment in streptozotocin-induced diabetic animals showed similar results. The data from this study indicate that hyperhexosemia-induced Akt/PKB activation may be an important mechanism leading to NF-kappaB and AP-1 activation and increased ECM protein synthesis in the organs affected by chronic diabetic complications.     

IFN-gamma-producing dendritic cells are important for priming of gut intraepithelial lymphocyte response against intracellular parasitic infection

The importance of intraepithelial lymphocytes (IEL) in immunoprotection against orally acquired pathogens is being increasingly recognized. Recent studies have demonstrated that Ag-specific IEL can be generated and can provide an important first line of defense against pathogens acquired via oral route. However, the mechanism involved in priming of IEL remains elusive. Our current study, using a microsporidial model of infection, demonstrates that priming of IEL is dependent on IFN-gamma-producing dendritic cells (DC) from mucosal sites. DC from mice lacking the IFN-gamma gene are unable to prime IEL, resulting in failure of these cells to proliferate and lyse pathogen-infected targets. Also, treatment of wild-type DC from Peyer's patches with Ab to IFN-gamma abrogates their ability to prime an IEL response against Encephalitozoon cuniculi in vitro. Moreover, when incubated with activated DC from IFN-gamma knockout mice, splenic CD8(+) T cells are not primed efficiently and exhibit reduced ability to home to the gut compartment. These data strongly suggest that IFN-gamma-producing DC from mucosal sites play an important role in the generation of an Ag-specific IEL response in the small intestine. To our knowledge, this report is the first demonstrating a role for IFN-gamma-producing DC from Peyer's patches in the development of Ag-specific IEL population and their trafficking to the gut epithelium.     

Salmonella induces a switched antibody response without germinal centers that impedes the extracellular spread of infection

T-dependent Ab responses are characterized by parallel extrafollicular plasmablast growth and germinal center (GC) formation. This study identifies that, in mice, the Ab response against Salmonella is novel in its kinetics and its regulation. It demonstrates that viable, attenuated Salmonella induce a massive early T-dependent extrafollicular response, whereas GC formation is delayed until 1 mo after infection. The extrafollicular Ab response with switching to IgG2c, the IgG2a equivalent in C57BL/6 mice, is well established by day 3 and persists through 5 wk. Switching is strongly T dependent, and the outer membrane proteins are shown to be major targets of the early switched IgG2c response, whereas flagellin and LPS are not. GC responses are associated with affinity maturation of IgG2c, and their induction is associated with bacterial burden because GC could be induced earlier by treating with antibiotics. Clearance of these bacteria is not a consequence of high-affinity Ab production, for clearance occurs equally in CD154-deficient mice, which do not develop GC, and wild-type mice. Nevertheless, transferred low- and high-affinity IgG2c and less efficiently IgM were shown to impede Salmonella colonization of splenic macrophages. Furthermore, Ab induced during the infection markedly reduces bacteremia. Thus, although Ab does not prevent the progress of established splenic infection, it can prevent primary infection and impedes secondary hemogenous spread of the disease. These results may explain why attenuated Salmonella-induced B cell responses are protective in secondary, but not primary infections.     

Vif counteracts a cyclophilin A-imposed inhibition of simian immunodeficiency viruses in human cells

Vif is a primate lentiviral accessory protein that is crucial for viral infectivity. Vif counteracts the antiviral activity of host deaminases such as APOBEC3G and APOBEC3F. We now report a novel function of African green monkey simian immunodeficiency virus (SIVagm) Vif that promotes replication of SIVagm in human cells lacking detectable deaminase activity. We found that cyclophilin A (CypA) was excluded from wild-type SIV particles but was efficiently packaged into vif-deficient SIVagm virions. The presence of CypA in vif-defective SIVagm was correlated with reduced viral replication. Infection of CypA knockout Jurkat cells or treatment of Jurkat cells with cyclosporine A eliminated the Vif-sensitive inhibition and resulted in replication profiles that were similar for wild-type and vif-deficient SIVagm. Importantly, the inhibitory effect of CypA was restricted to virus-producing cells and was TRIM5alpha independent. The abilities of SIVagm Vif to inhibit encapsidation of CypA and to increase viral infectivity were shared by rhesus macaque SIV Vif and thus seem to be general properties of SIV Vif proteins. Exclusion of CypA from SIVagm particles was not associated with intracellular degradation, suggesting a mode of Vif action distinct from that proposed for APOBEC3G. This is the first report of a novel vif-sensitive antiviral activity of human CypA that may limit zoonotic transmission of SIV and the first demonstration of CypA encapsidation into a virus other than human immunodeficiency virus type 1.     

A constitutive, transient receptor potential-like Ca2+ influx pathway in presynaptic nerve endings independent of voltage-gated Ca2+ channels and Na+/Ca2+ exchange

Calcium levels in the presynaptic nerve terminal are altered by several pathways, including voltage-gated Ca(2+) channels, the Na(+)/Ca(2+) exchanger, Ca(2+)-ATPase, and the mitochondria. The influx pathway for homeostatic control of [Ca(2+)](i) in the nerve terminal has been unclear. One approach to detecting the pathway that maintains internal Ca(2+) is to test for activation of Ca(2+) influx following Ca(2+) depletion. Here, we demonstrate that a constitutive influx pathway for Ca(2+) exists in presynaptic terminals to maintain internal Ca(2+) independent of voltage-gated Ca(2+) channels and Na(+)/Ca(2+) exchange, as measured in intact isolated nerve endings from mouse cortex and in intact varicosities in a neuronal cell line using fluorescence spectroscopy and confocal imaging. The Mg(2+) and lanthanide sensitivity of the influx pathway, in addition to its pharmacological and short hairpin RNA sensitivity, and the results of immunostaining for transient receptor potential (TRP) channels indicate the involvement of TRPC channels, possibly TRPC5 and TRPC1. This constitutive Ca(2+) influx pathway likely serves to maintain synaptic function under widely varying levels of synaptic activity.     

A model-based parametric study of impact force during running

This paper deals with the impact force during foot-ground impact activities such as the running. A previously developed model is used for this study. The model is a lumped-parameter one consisting of four masses connected to each other via linear springs and viscous dampers. A shoe-specific nonlinear function is used for representation of the ground reaction force. The authors have previously showed that the previous version of the model as well as its simulation is incorrect. This paper slightly modifies the previous model so as it is able to produce results in agreement with the experiments. Then, the modified model is simulated for two typical shoe types. A parametric study is also conducted. The parametric study concerns with the effects of masses, mass ratios, stiffness constants, and damping coefficients on the dynamics of the impact. It is shown that the impact forces increase as the rigid and wobbling masses increase. However, the increase in the impact forces is not the same for all the masses. It is found that the impact force increases as the touchdown velocities increase. Simulations imply that the variations of the damping coefficients result in larger variations of the impact force compared to the stiffness. The effect of the variation of gravity on the simulated impact force is also explored. It is concluded that both the first and the second peaks of the impact force are increased with gravity. An in-depth discussion is included to compare results of the current paper with results of other investigators.