In vivo Kinetic Biodistribution of Nano‐Sized Outer Membrane Vesicles Derived from Bacteria

Evaluation of kinetic distribution and behaviors of nanoparticles in vivo provides crucial clues into their roles in living organisms. Extracellular vesicles are evolutionary conserved nanoparticles, known to play important biological functions in intercellular, inter‐species, and inter‐kingdom communication. In this study, the first kinetic analysis of the biodistribution of outer membrane vesicles (OMVs)—bacterial extracellular vesicles—with immune‐modulatory functions is performed. OMVs, injected intraperitoneally, spread to the whole mouse body and accumulate in the liver, lung, spleen, and kidney within 3 h of administration. As an early systemic inflammation response, increased levels of TNF‐α and IL‐6 are observed in serum and bronchoalveolar lavage fluid. In addition, the number of leukocytes and platelets in the blood is decreased. OMVs and cytokine concentrations, as well as body temperature are gradually decreased 6 h after OMV injection, in concomitance with the formation of eye exudates, and of an increase in ICAM‐1 levels in the lung. Following OMV elimination, most of the inflammatory signs are reverted, 12 h post‐injection. However, leukocytes in bronchoalveolar lavage fluid are increased as a late reaction. Taken together, these results suggest that OMVs are effective mediators of long distance communication in vivo.     

Measurement and numerical simulation of three‐dimensional fiber orientation states in injection‐molded short‐fiber‐reinforced plastics

To determine three‐dimensional fiber orientation states in injection‐molded short‐fiber composites, a confocal laser scanning microscope (CLSM) is used. Since the CLSM optically sections the specimen, more than two images of the cross sections on and below the surface of the composite can be obtained. Three‐dimensional fiber orientation states can be determined by using geometric parameters of fiber images obtained from two parallel cross sections. For experiments, carbon‐fiber‐reinforced polystyrene is examined by the CLSM and geometric parameters of fibers on each cross‐sectional plane are measured by an image analysis. In order to describe fiber orientation states compactly, orientation tensors are determined at different positions of the prepared specimen. Three‐dimensional orientation states are obtained without any difficulty by determining the out‐of‐plane angles utilizing fiber images on two parallel planes acquired by the CLSM. Orientation states are different at different positions and show the shell–core structure along the thickness of the specimen. Fiber orientation tensors are predicted by a numerical analysis and the numerically predicted orientation states show good agreement with measured ones. However, some differences are found at the end of cavity. They may result from the fountain flow effects, which are not considered in the numerical analysis. © 2003 Wiley Periodicals, Inc. J Appl Polym Sci 88: 500–509, 2003     

CMV colitis masquerading as colon cancer--an unusual presentation of acquired immunodeficiency syndrome

To determine whether there is any correlation between sudden decrease in barometric pressure and onset of labor, a non-experimental, retrospective study at a 948-bed tertiary care hospital was done. Pregnant patients of 36 weeks gestation or more who presented with spontaneous onset of labor during the 48 hours surrounding the 12 occurrences of significant drop in barometric pressure in 1992 were included in the study. Significantly more occurrences of onset of labor were identified in the 24 hours after a drop in barometric pressure than were identified in the 24 hours prior to the drop in barometric pressure (P < 0.05). Therefore, the overall number of labor onsets increased in the 24 hours following a significant drop in barometric pressure.     

Mutational analysis of the three cysteines and active-site aspartic acid 103 of ketosteroid isomerase from Pseudomonas putida biotype B

In order to clarify the roles of three cysteines in ketosteroid isomerase (KSI) from Pseudomonas putida biotype B, each of the cysteine residues has been changed to a serine residue (C69S, C81S, and C97S) by site-directed mutagenesis. All cysteine mutations caused only a slight decrease in the k(cat) value, with no significant change of Km for the substrate. Even modification of the sulfhydryl group with 5,5'-dithiobis(2-nitrobenzoic acid) has almost no effect on enzyme activity. These results demonstrate that none of the cysteines in the KSI from P. putida is critical for catalytic activity, contrary to the previous identification of a cysteine in an active-site-directed photoinactivation study of KSI. Based on the three-dimensional structures of KSIs with and without dienolate intermediate analog equilenin, as determined by X-ray crystallography at high resolution, Asp-103 was found to be located within the range of the hydrogen bond to the equilenin. To assess the role of Asp-103 in catalysis, Asp-103 has been replaced with either asparagine (D103N) or alanine (D103A) by site-directed mutagenesis. For D103A mutant KSI there was a significant decrease in the k(cat) value: the k(cat) of the mutant was 85-fold lower than that of the wild-type enzyme; however, for the D103N mutant, which retained some hydrogen bonding capability, there was a minor decrease in the k(cat) value. These findings support the idea that aspartic acid 103 in the active site is an essential catalytic residue involved in catalysis by hydrogen bonding to the dienolate intermediate.     

Intercellular communication between dorsal root ganglion cells and colonic smooth muscle cells in vitro

This study demonstrates that the use of high field 1H NMR spectroscopy permits individual detection of phosphatidylcholine and sphingomyelin molecules at the surface of native low density lipoprotein (LDL) particles. Distinct behaviour was observed for the choline head group -N(CH3)3 resonances of these different phospholipids revealing preferential immobilisation for phosphatidylcholine. This suggests the existence of reversible and irreversible phosphatidylcholine-apolipoprotein B interactions and is consistent with microdomain formation at the surface monolayer of LDL. The novel resonance assignment and results show that 1H NMR can provide efficient and practical means for future studies on the structure and dynamics at the LDL surface.     

LP-BM5 virus causes changes in lactic dehydrogenase and oxygen radical production in splenocytes of C57BL/6J mice

The LP-BM5 murine leukemia virus causes acquired immunodeficiency syndrome in C57BL/6J mice (MAIDS), similar to that of AIDS in humans. The objective of this study was to determine the effect of LP-BM5 viral infection on cellular activation and membrane integrity of splenocytes. Oxidative burst in splenocytes in response to exposure to PMA (20 microg/ml) was significantly higher (p<.02) in infected than in control mice at two weeks post-infection using luminol-enhanced chemiluminescence. By 13 weeks post-infection superoxide anion production in infected mice was significantly lower when compared to controls coinciding with decreased proliferative response to mitogens. The extent of cell membrane damage as indicated by lactic dehydrogenase (LDH) activity in serum was significantly higher in infected than in control mice (p<.001). The results from this study suggests that LP-BM5 virus causes an initial stimulation of cellular activity followed by a decreased cell activation characterized by decreased proliferation of splenocytes and decreased oxygen radical production. Decreased cell membrane integrity indicated by increased LDH activity may partly be responsible for these changes.     

Antiserum against Escherichia coli J5 contains antibodies reactive with outer membrane proteins of heterologous gram-negative bacteria

The binding of IgG in antiserum to Escherichia coli J5 to the surface of Enterobacteriaceae and to cell wall fragments released from serum-exposed bacteria was studied in a search for potentially protective epitopes other than lipopolysaccharide (LPS). IgG titers to multiple heterologous gram-negative smooth bacteria increased following incubation of the bacteria in serum and decreased following absorption with serum-exposed heterologous bacteria. IgG eluted from absorbing bacteria bound to at least three conserved bacterial outer membrane proteins (OMPs), but not LPS, as assessed by immunoblotting. The same OMPs were present in LPS-containing macromolecular cell wall fragments released by incubation of heterologous gram-negative bacteria in human serum. Part of the protection offered by J5 antiserum could be from binding of IgG to conserved OMPs at the bacterial surface or to OMPs in cell-wall fragments released from dying bacteria.