Effects of gradual expansign on arterial stenosis

The blood flow through an arterial stenosis can theoretically be increased by gradual expansion of the stenosis. This hypothesis was tested by comparing the flow through abruptly expanding and gradually expanding stenosed plastic conduits. The conduits were tested in non-pulsatile flow with water and bank blood and in pulsatile flow in dog's descending aorta. It was found that:

1. (a)
   The pressure drop in arterial stenoses is larger than that predicted by non-pulsatile theory.     

Disruption of the Cytokeratin Cytoskeleton and Inhibition of Hepatocytic Autophagy by Okadaic Acid

To learn whether autophagy might be dependent on any of the major cytoskeletal elements, the effect of various cytoskeleton inhibitors on autophagy and cytoskeletal organization was studied in isolated rat hepatocytes. Autophagy, measured as the sequestration of endogenous lactate dehydrogenase, was completely inhibited in isolated rat hepatocytes by the protein phosphatase inhibitor okadaic acid (30 nM). Only small effects were seen with vinblastine (10 μM) or cytochalasin D (10 μM). Indirect immunofluorescence microscopy with antibody to a 55-kDa cytokeratin, corresponding to human cytokeratin 8 (CK8), revealed that whereas control cells contained a well-organized network of cytokeratin intermediate filaments, okadaic acid disrupted this network into small spherical aggregates. Treatment with cytochalasin D or vinblastine, which disrupt microfilaments and microtubules, respectively, had no detectable effect on the cytokeratin filament distribution. Neither the microtubule network (detected by indirect immunofluorescence with antibodies against α- and β-tubulin) nor the actin microfilament network (detected by rhodamine-palloidin) was disrupted by okadaic acid. Naringin (100 μM), a putative protein kinase-inhibitory flavonoid, offered complete protection against the autophagy-inhibitory and cytokeratin-disruptive effects of okadaic acid. Two other flavonoids, genistein (100 μM) and prunin (100 μM) as well as KN-62 (10 μM), a specific inhibitor of Ca²⁺/calmodulin-dependent kinase II), likewise displayed a good ability to protect against the effect of okadaic acid upon cytokeratin organization, while no such protection was seen with H-89 (20 μM), an inhibitor of the cyclic nucleotide-dependent protein kinases, or with H-7 (100 μM), which in addition inhibits protein kinase C. The results suggest that the cytokeratin cytoskeleton of hepatocytes is subject to rapid control by phosphorylation and dephosphorylation and that cytokeratin filaments may somehow be involved in the autophagic process.     

Structures and activities of widely conserved small prokaryotic aminopeptidases-P clarify classification of M24B peptidases

M24B peptidases cleaving Xaa-Pro bond in dipeptides are prolidases whereas those cleaving this bond in longer peptides are aminopeptidases-P. Bacteria have small aminopeptidases-P (36-39 kDa), which are diverged from canonical aminopeptidase-P of Escherichia coli (50 kDa). Structure-function studies of small aminopeptidases-P are lacking. We report crystal structures of small aminopeptidases-P from E. coli and Deinococcus radiodurans, and report substrate-specificities of these proteins and their ortholog from Mycobacterium tuberculosis. These are aminopeptidases-P, structurally close to small prolidases except for absence of dipeptide-selectivity loop. We noticed absence of this loop and conserved arginine in canonical archaeal prolidase (Maher et al., Biochemistry. 43, 2004, 2771-2783) and questioned its classification. Our enzymatic assays show that this enzyme is an aminopeptidase-P. Further, our mutagenesis studies illuminate importance of DXRY sequence motif in bacterial small aminopeptidases-P and suggest common evolutionary origin with human XPNPEP1/XPNPEP2. Our analyses reveal sequence/structural features distinguishing small aminopeptidases-P from other M24B peptidases.     

Impaired secretion of IL-10 by T cells from patients with common variable immunodeficiency--involvement of protein kinase A type I

Common variable immunodeficiency (CVID) is a heterogeneous group of B cell deficiency syndromes. T cell abnormalities are present in a high proportion of patients with CVID, suggesting impaired T cell-mediated stimulation of B cells. Based on the importance of IL-10 for B cell function and the involvement of the cAMP/protein kinase A type I (PKAI) system in IL-10 synthesis, we examined IL-10 secretion in T cells from CVID patients and controls, particularly focusing on possible modulatory effects of the cAMP/PKAI system. Our main findings were: 1) anti-CD3 and anti-CD3/anti-CD28 activated T cells from CVID patients secreted less IL-10 than healthy controls. This defect was not related to varying proportions of T cell subsets (e.g., CD4(+)/CD8(+), CD45RA(+)/RO(+), or CD28(-) T cells); 2) PKAI activation through the cAMP agonist 8-CPT-cAMP markedly inhibited IL-10 secretion from T cells through CD3 and CD28 activation in both patients and controls, but the sensitivity for cAMP-dependent inhibition was increased in CVID; 3) selective PKAI inhibition by Rp-8-Br-cAMPS markedly increased IL-10 secretion in anti-CD3 and anti-CD3/anti-CD28-stimulated T cells in both patients and controls. Even at the lowest concentrations of Rp-8-Br-cAMPS, IL-10 secretion in CVID patients reached levels comparable to those in controls. Our findings suggest impaired secretion of IL-10 by T cells from CVID patients, suggesting a possible link between T cell deficiency and impaired B cell function in CVID. The involvement of the cAMP/PKAI system in this defect suggests a novel target for therapeutic immunomodulation in CVID.     

Reduced immunocompetence and cost of reproduction in common eiders

Immunocompetence may be especially important in long-lived species where infectious organisms may have detrimental effects upon future reproductive value of hosts. The resource demand for immunocompetence may reduce resource availability for reproduction and a trade-off between these traits has therefore been proposed. Capital breeders, such as the common eider (Somateria mollissima), rely upon accumulated body reserves during reproduction. Eiders lose more than 40% of pre-breeding body mass during egglaying and incubation and many females abandon their ducklings to other females after hatching. Results from our observational study show that levels of leukocytes (i.e., lymphocytes, heterophils and heterophil/lymphocyte ratio) are not related to body mass early in the incubation period. However, eider females with low initial body mass showed signs of immunosuppression (i.e., decreased late levels of lymphocytes) and increased response towards stressors (i.e., increased heterophil/lymphocyte ratio) later in the incubation period. Moreover, females with low lymphocyte levels more frequently abandoned their brood, and females abandoning young had an increased return rate to the next breeding season. However, among brood abandoning females return rate was lower for the females with low lymphocyte levels. These results suggest that immunosuppression, as indicated by low lymphocyte levels, is a reproductive cost that may be partly compensated for by abandoning young.     

Use of Real-Time Quantitative PCR for the Analysis of φLC3 Prophage Stability in Lactococci

Bacteriophages are a common and constant threat to proper milk fermentation. It has become evident that lysogeny is widespread in lactic acid bacteria, and in this work the temperate lactococcal bacteriophage LC3 was used as a model to study prophage stability in lactococci. The stability was analyzed in six LC3 lysogenic Lactococcus lactis subsp. cremoris host strains when they were growing at 15 and 30°C. In order to perform these analyses, a real-time PCR assay was developed. The stability of the LC3 prophage was found to vary with the growth phase of its host L. lactis IMN-C1814, in which the induction rate increased during the exponential growth phase and reached a maximum level when the strain was entering the stationary phase. The maximum spontaneous induction frequency of the LC3 prophage varied between 0.32 and 9.1% (28-fold) in the six lysogenic strains. No correlation was observed between growth rates of the host cells and the spontaneous prophage induction frequencies. Furthermore, the level of extrachromosomal phage DNA after induction of the prophage varied between the strains (1.9 to 390%), and the estimated burst sizes varied up to eightfold. These results show that the host cells have a significant impact on the lytic and lysogenic life styles of temperate bacteriophages. The present study shows the power of the real-time PCR technique in the analysis of temperate phage biology and will be useful in work to reveal the impact of temperate phages and lysogenic bacteria in various ecological fields.