Pre-existing glomerular immune complexes induce polymorphonuclear cell recruitment through an Fc receptor-dependent respiratory burst: potential role in the perpetuation of immune nephritis

In immune complex (IC) diseases, FcR are essential molecules facilitating polymorphonuclear cell (PMN) recruitment and effector functions at the IC site. Although FcR-dependent initial tethering and FcR/integrin-dependent PMN accumulation were postulated, their underlying mechanisms remain unclear. We here addressed potential mechanisms involved in PMN recruitment in acute IC glomerulonephritis (nephrotoxic nephritis). Since some renal cells may be recruited from bone marrow (BM) lineages, reconstitution studies with BM chimeras and PMN transfer between wild-type (WT) and FcR-deficient mice (gamma(-/-)) were performed. Severe glomerular damage was induced in WT and W gamma chimeras (BM from WT to irradiated gamma(-/-)), while it was absent in gamma(-/-) and gamma W chimeras (gamma(-/-) BM to WT). Moreover, WT PMN transfer, but not gamma(-/-) PMN, reconstituted the disease in gamma(-/-), indicating that FcR on resident cells is not a prerequisite for PMN recruitment in this disease. Surprisingly, transferred WT PMN were recruited coincidentally with NF-kappa B activation and TNF-alpha overexpression even in glomeruli with preformed IC (nephrotoxic Ab administered 3 days previously), suggesting that PMN can initially be recruited via its own FcR without previous chemoattractant release. Furthermore, H(2)O(2) inhibition by catalase attenuated the acute WT PMN recruitment and the induction of NF-kappa B and TNF-alpha much more than integrin (CD18) blockade, indicating a role for the respiratory burst before integrin-dependent accumulation. In coculture experiments with IC-stimulated PMN and glomeruli, PMN caused acute glomerular TNF-alpha expression predominantly via FcR-mediated H(2)O(2) production. In conclusion, glomerular IC, even preformed, can cause PMN recruitment and injury through PMN FcR-mediated respiratory burst during initial PMN tethering to IC.     

Recombinant bovine herpesvirus-1 expressing p23 protein of Cryptosporidium parvum induces neutralizing antibodies in rabbits

In order to develop a vaccine against cryptosporidiosis in cattle, we constructed a recombinant bovine herpesvirus-1 (BHV-1) expressing an immunodominant surface protein, p23, of Cryptosporidium parvum sporozoites. In the recombinant virus, the p23 gene under the control of a CAG promoter and a gene coding for an enhanced green fluorescent protein were integrated into the gG gene of BHV-1. Despite a low frequency of homologous recombination, cloning of the recombinants was easy because of the specific fluorescence of the plaques formed by recombinants. These plaques were among the plaques of the nonfluorescent parental virus. All clones selected for fluorescence also contained the p23 gene. In MDBK cells infected with the recombinant BHV-1, the antibody against the p23 protein recognized the p23 protein as an approximately 23-kDa specific band in Western blotting analysis. Rabbits immunized with the recombinant produced IgG against the p23 protein. It was also demonstrated that the sera of immunized rabbits reduced infection of C. parvum sporozoites in HCT-8 cells. The serum of an immunized rabbit reduced infection compared with the normal rabbit serum control. These results indicate that the recombinant BHV-1 induces neutralizing antibodies in rabbits.     

Sphingosine kinase type 1 induces G12/13-mediated stress fiber formation,yet promotes growth and survival independent of G protein-coupled receptors

Sphingosine 1-phosphate (S1P) is the ligand for a family of specific G protein-coupled receptors (GPCRs) that regulate a wide variety of important cellular functions, including growth, survival, cytoskeletal rearrangements, and cell motility. However, whether it also has an intracellular function is still a matter of great debate. Overexpression of sphingosine kinase type 1, which generated S1P, induced extensive stress fibers and impaired formation of the Src-focal adhesion kinase signaling complex, with consequent aberrant focal adhesion turnover, leading to inhibition of cell locomotion. We have dissected biological responses dependent on intracellular S1P from those that are receptor-mediated by specifically blocking signaling of Galphaq, Galphai, Galpha12/13, and Gbetagamma subunits, the G proteins that S1P receptors (S1PRs) couple to and signal through. We found that intracellular S1P signaled "inside out" through its cell-surface receptors linked to G12/13-mediated stress fiber formation, important for cell motility. Remarkably, cell growth stimulation and suppression of apoptosis by endogenous S1P were independent of GPCRs and inside-out signaling. Using fibroblasts from embryonic mice devoid of functional S1PRs, we also demonstrated that, in contrast to exogenous S1P, intracellular S1P formed by overexpression of sphingosine kinase type 1 promoted growth and survival independent of its GPCRs. Hence, exogenous and intracellularly generated S1Ps affect cell growth and survival by divergent pathways. Our results demonstrate a receptor-independent intracellular function of S1P, reminiscent of its action in yeast cells that lack S1PRs.     

A femoral neck fracture model in rabbits

A technique was developed to create a reproducible femoral neck fracture in vitro using 5-month-old JW/CSK series male rabbits. Force attenuation of a newly developed damping material was also evaluated using this model. Ten pairs of the femora with smaller deviations in length and weight were harvested and cleaned of soft tissue. Either a right or left of each pair of the specimens was randomly selected and put into either the control or the experimental group, both of which contained equal numbers of the right and left femora. The specimens were attached to an L-shaped plate and embedded in a resin from the proximal diaphysis to the distal end so as to maintain a consistent position of the femora. They were mounted and fixed on a pedestal slanted in the coronal plane at 20 degrees. The impact load testing was conducted using an impact mallet dropped from a height of 3 cm. The impact load was applied onto the femoral head. To the specimens in the experimental group, attenuated impact forces were loaded through the damping material, but those in the control group were subjected to forces directly transmitted without the material. All the impact testing was performed in a temperature and humidity controlled chamber. All of the femoral specimens exposed to the direct impact forces (controlled group) sustained fracture at the neck. The fracture line passed from the base of the femoral head laterally and to the calcar area just proximal to the minor trochanter medially. The location of each fracture line was almost identical among the specimens. None of the specimens that were exposed to the impact force through the damping material (experimental group) sustained fracture macroscopically and roentgenographically.     

Mouse lymphoma thymidine kinase gene mutation assay: International Workshop on Genotoxicity Tests Workgroup report--Plymouth, UK 2002

The Mouse Lymphoma Assay (MLA) Workgroup of the International Workshop on Genotoxicity Tests (IWGT) met on June 28th and 29th, 2002, in Plymouth, England. This meeting of the MLA group was devoted to discussing the criteria for assay acceptance and appropriate approaches to data evaluation. Prior to the meeting, the group conducted an extensive analysis of data from both the microwell and soft agar versions of the assay. For the establishment of criteria for assay acceptance, 10 laboratories (6 using the microwell method and 4 using soft agar) provided data on their background mutant frequencies, plating efficiencies of the negative/vehicle control, cell suspension growth, and positive control mutant frequencies. Using the distribution curves generated from this data, the Workgroup reached consensus on the range of values that should be used to determine whether an individual experiment is acceptable. In order to establish appropriate approaches for data evaluation, the group used a number of statistical methods to evaluate approximately 400 experimental data sets from 10 laboratories entered into a database created for the earlier MLA Workshop held in New Orleans [Environ. Mol. Mutagen. 40 (2002) 292]. While the Workgroup could not, during this meeting, make a final recommendation for the evaluation of data, a general strategy was developed and the Workgroup members agreed to evaluate this new proposed approach using their own laboratory data. This evaluation should lead to a consensus global approach for data evaluation in the near future.     

L-cysteine administration prevents liver fibrosis by suppressing hepatic stellate cell proliferation and activation

Recent studies showed that the function of some amino acids is not only nutritional but also pharmacological. However, the effects of amino acids on liver fibrosis and hepatic stellate cell (HSC) remain unclear. In this research, as a result of screening of amino acids using liver fibrosis induced by DMN administration, L-cysteine was selected as a suppressor of liver fibrosis. Furthermore, the number of activated HSCs, which increased in the fibrotic liver after DMN administration, was decreased in L-cysteine-fed rats. Treatment of freshly isolated HSCs with L-cysteine resulted in inhibition of the increase in smooth muscle alpha-actin (alphaSMA) expression by HSCs and BrdU incorporation into the activated HSCs. These findings suggest that L-cysteine is effective against liver fibrosis. The mechanism of inhibition of fibrosis in the liver is surmized to be direct inhibition of activated HSC proliferation and HSC transformation by L-cysteine.     

Activation of hydrogen peroxide in horseradish peroxidase occurs within approximately 200 micro s observed by a new freeze-quench device

To observe the formation process of compound I in horseradish peroxidase (HRP), we developed a new freeze-quench device with approximately 200 micro s of the mixing-to-freezing time interval and observed the reaction between HRP and hydrogen peroxide (H(2)O(2)). The developed device consists of a submillisecond solution mixer and rotating copper or silver plates cooled at 77 K; it freezes the small droplets of mixed solution on the surface of the rotating plates. The ultraviolet-visible spectra of the sample quenched at approximately 1 ms after the mixing of HRP and H(2)O(2) suggest the formation of compound I. The electron paramagnetic resonance spectra of the same reaction quenched at approximately 200 micro s show a convex peak at g = 2.00, which is identified as compound I due to its microwave power and temperature dependencies. The absence of ferric signals in the electron paramagnetic resonance spectra of the quenched sample indicates that compound I is formed within approximately 200 micro s after mixing HRP and H(2)O(2). We conclude that the activation of H(2)O(2) in HRP at ambient temperature completes within approximately 200 micro s. The developed device can be generally applied to investigate the electronic structures of short-lived intermediates of metalloenzymes.     

Elongational flow studies on conformational change in DNA induced by DNA-binding protein HU

Interaction of DNA-binding protein HU from Bacillus stearothermophilis (HUBst) with coliphage T2 DNA was investigated by observing an elongational flow-induced birefringence, Deltan, of a T2-phage DNA aqueous solution at various HU concentrations. Localized flow birefringence was observed in the pure elongational flow region, and the strain rate dependence of Deltan had a critical strain rate epsilon;(c) for the appearance of flow birefringence at all of the HU concentrations examined, indicating that a coil-stretch transition occurred at epsilon;(c) in each DNA-HU system. For strain rates larger than epsilon;(c), Deltan increased rapidly and then gradually, approaching a plateau value. The value of epsilon;(c) increased with an increase in HU concentration. Analysis based on the relationship between epsilon; (c) and the Rouse-Zimm relaxation time revealed that the increase in epsilon;(c)with increase in HU can be explained by the decrease in the size of the DNA-HU complex. The plateau birefringence value, Deltan(p), decreased at small HU concentrations but did not change at larger HU concentrations. Considering that Deltan(p) is related to the orientational order parameter of segments, it was concluded that there were at least two stages in the process of compaction of DNA induced by HU.     

Structure and physicochemical properties of starches from kidney bean seeds at immature,premature and mature stages of development

Starches from kidney bean (Phaseolus vulgaris L. cv. Toramame) seeds at the immature, premature, mature stages of development were examined. The starch content increased from 94, 219 to 265 mg per seed. Starches showed the C(a)-crystalline type composed of small (<5 micrometer) and large (10-35 micrometer) granules, with the large granules largely increasing with maturity. The amylose content increased from 21, 26 to 27%, and rapid viscograms and DSC thermograms suggested that the mature-stage starch was gelatinized with ease. The amylose increased in size from DPn 820, 1000 to 1080 and a number of chains per molecule (NC) from 3.3, 4.2 to 4.5. The branched amylose was a minor component (11-18% by mole) with NC 20-22. The amylopectin was similar in CL (23), beta-amylolysis limit (59%), and chain-length distribution, but reduced in size (DPn 17,100-5270) and increased in content of phosphorus (114-174 ppm) with an increase in the amount of phosphorus linked to C-6 of the glucose residue (8-66%).