Defects in somite formation in lunatic fringe-deficient mice

Segmentation in vertebrates first arises when the unsegmented paraxial mesoderm subdivides to form paired epithelial spheres called somites. The Notch signalling pathway is important in regulating the formation and anterior-posterior patterning of the vertebrate somite. One component of the Notch signalling pathway in Drosophila is the fringe gene, which encodes a secreted signalling molecule required for activation of Notch during specification of the wing margin. Here we show that mice homozygous for a targeted mutation of the lunatic fringe (Lfng) gene, one of the mouse homologues of fringe, have defects in somite formation and anterior-posterior patterning of the somites. Somites in the mutant embryos are irregular in size and shape, and their anterior-posterior patterning is disturbed. Marker analysis revealed that in the presomitic mesoderm of the mutant embryos, sharply demarcated domains of expression of several components of the Notch signalling pathway are replaced by even gradients of gene expression. These results indicate that Lfng encodes an essential component of the Notch signalling pathway during somitogenesis in mice.     

Norepinephrine loss selectively enhances chronic nigrostriatal dopamine depletion in mice and rats

A total of 3,700 Pseudomonas aeruginosa isolates were collected from 17 general hospitals in Japan from 1992 to 1994. Of these isolates, 132 carbapenem-resistant strains were subjected to DNA hybridization analysis with the metallo-beta-lactamase gene (blaIMP)-specific probe. Fifteen strains carrying the metallo-beta-lactamase gene were identified in five hospitals in different geographical areas. Three strains of P. aeruginosa demonstrated high-level imipenem resistance (MIC, > or = 128 micrograms/ml), two strains exhibited low-level imipenem resistance (MIC, < or = 4 micrograms/ml), and the rest of the strains were in between. These results revealed that the acquisition of a metallo-beta-lactamase gene alone does not necessarily confer elevated resistance to carbapenems. In several strains, the metallo-beta-lactamase gene was carried by large plasmids, and carbapenem resistance was transferred from P. aeruginosa to Escherichia coli by electroporation in association with the acquisition of the large plasmid. Southern hybridization analysis and genomic DNA fingerprinting profiles revealed different genetic backgrounds for these 15 isolates, although considerable similarity was observed for the strains isolated from the same hospital. These findings suggest that the metallo-beta-lactamase-producing P. aeruginosa strains are not confined to a unique clonal lineage but proliferated multifocally by plasmid-mediated dissemination of the metallo-beta-lactamase gene in strains of different genetic backgrounds. Thus, further proliferation of metallo-beta-lactamase-producing strains with resistance to various beta-lactams may well be inevitable in the future, which emphasizes the need for early recognition of metallo-beta-lactamase-producing strains, rigorous infection control, and restricted clinical use of broad-spectrum beta-lactams including carbapenems.     

The integrin ligand echistatin prevents bone loss in ovariectomized mice and rats

Integrins that bind RGD (arginine-glycine-aspartic acid) containing peptides, especially the vitronectin receptor alpha(v)beta3, have been implicated in the regulation of osteoclast function. Echistatin, an RGD-containing snake venom peptide with high affinity for beta3 integrins, as well as nonpeptide RGD mimetics, were shown to inhibit osteoclastic bone resorption in vitro and in vivo. To evaluate the role of RGD-binding integrins in bone metabolism, we examined by several methods the effects of echistatin on ovariectomy (OVX)-induced bone loss in mice and rats. First, we confirmed that echistatin binds in vitro with high affinity (Kd, 0.5 nM) to alpha(v)beta3 integrin purified from human placenta and established a competitive binding assay to measure echistatin concentrations in serum. We find that echistatin infused for 2 or 4 weeks at 0.36 microg/h x g body weight (approximately 50 nmol/day x mouse) completely prevents OVX-induced cancellous bone loss in the distal femora of ovariectomized mice. Echistatin has no effect on uterine weight, body weight, and femoral length changes induced by OVX, nor does it cause any apparent changes in major organs other than bone. In OVX rats, echistatin infusion at 0.26 microg/h x g for 4 weeks effectively prevents bone loss, evaluated by dual energy x-ray absorptiometry of the femur, by femoral ash weight, and by bone histomorphometry of the proximal tibia. At effective serum concentrations of 20-30 nM, measured at the end of the infusion period, echistatin maintains histomorphometric indices of bone turnover at control levels but does not decrease osteoclast surface. In conclusion, these results provide in vivo evidence, at the level of bone histology, that RGD-binding integrins, probably alpha(v)beta3, play a rate-limiting role in osteoclastic bone resorption and suggest a therapeutic potential for integrin ligands in the suppression of bone loss.     

Reduction in amyloid A amyloid formation in apolipoprotein-E-deficient mice

Apolipoproteins have been implicated in the formation of amyloid fibrils. Recent studies have demonstrated that apolipoprotein E (apoE), alone or in combination with apolipoprotein J (apoJ), and other lipoproteins appear to enhance deposition of amyloid fibrils both in systemic and cerebral amyloids, especially Alzheimer's disease (AD). ApoE enhanced the ability of the amyloid beta-protein (1-40) fragment (A beta) to form fibrils in vitro, with apoE4 promoting the greatest fibril formation. ApoE was found associated with both human and mouse amyloid A (AA) deposits. To define the role of apoE in vivo, we utilized mice lacking the apoE gene by gene targeting. We used the AA model in mice to characterize the function of the apoE protein in amyloid fibrillogenesis. ApoE-deficient mice exhibited a decrease in deposition of AA when compared with heterozygous mutant or wild-type animals. In addition, apoE-deficient mice that were injected with an adenovirus that expressed the human apoE3 gene had restored AA deposition and the apoE was associated with the AA fibrils. These results are agreement with the in vitro studies using the beta-peptide and suggest that apoE is not essential for amyloid fibrillogenesis but can promote the development of amyloid deposition.     

Dissociation between bone resorption and bone formation in osteopenic transgenic mice

Bone mass is maintained constant in vertebrates through bone remodeling (BR). BR is characterized by osteoclastic resorption of preexisting bone followed by de novo bone formation by osteoblasts. This sequence of events and the fact that bone mass remains constant in physiological situation lead to the assumption that resorption and formation are regulated by each other during BR. Recent evidence shows that cells of the osteoblastic lineage are involved in osteoclast differentiation. However, the existence of a functional link between the two activities, formation and resorption, has never been shown in vivo. To define the role of bone formation in the control of bone resorption, we generated an inducible osteoblast ablation mouse model. These mice developed a reversible osteopenia. Functional analyses showed that in the absence of bone formation, bone resorption continued to occur normally, leading to an osteoporosis of controllable severity, whose appearance could be prevented by an antiresorptive agent. This study establishes that bone formation and/or bone mass do not control the extent of bone resorption in vivo.     

IkappaBalpha overexpression delays tumor formation in v-rel transgenic mice

Glutamate is the major excitatory neurotransmitter in the retina, but excessive stimulation of its receptors leads to widespread neuronal stress and death. Both growth factors and gangliosides display important influences on responses to neuronal injury and degeneration. In this study, we have investigated the potential protective effects of two well characterized growth factors, epidermal and basic fibroblast growth factor (EGF and bFGF respectively), and the monosialoganglioside GM1, on cultured rat retinal neurons submitted to toxic levels of excitatory amino acids. Application of 1 mM glutamic acid reduced global neuronal viability by 80% when compared to control untreated cultures, whereas treatment with the glutamic acid agonist kainic acid (1 mM) led to specific, large decreases (75% reduction) in amacrine cell numbers. 24 h pretreatment with either EGF or bFGF (500 pM each) prevented the majority of excitatory amino acid-induced neuronal death, whereas similar treatment with 10(-5) M GM1 did not block neuronal degeneration. These findings demonstrate that EGF and bFGF act as neuroprotective agents against retinal excitotoxicity in vitro, whereas ganglioside GM1 is not effective in this particular paradigm.     

Dislocation network formation and coherency loss around gamma- prime precipitates in a nickel- base superalloy

A study of the loss of coherency suffered by gamma-prime precipitates after aging or through plastic deformation was carried out in a superalloy, IN 713C* Selected aging treatments were employed so as to observe different stages of coherency loss. Examination using transmission electron microscopy reveals that itinerant or matrix dislocations, generated either by internal stresses (carbides,etc.) or through the usual processes of general plastic deformation, rearrange themselves by cross-slip and climb around gamma-prime precipitates to produce misfit dislocation networks. Along with the network formation, the precipitate morphology changes to maximize the misfit accommodation. The final dislocation and precipitate configurations are consistent with the Lasalmonie and Strudel model.     

Ischemia-induced neuronal cell loss is associated with loss of atypical angiotensin type-1 receptor expression in the gerbil hippocampal formation

The hippocampal formation of Mongolian gerbils expresses high amounts of atypical angiotensin II type-1 receptors. We studied the expression of these receptors by in situ hybridization using specific [35S]-labeled riboprobes and by receptor autoradiography using [125I]Sarcosine1-angiotensin II. Angiotensin II receptor mRNA was found in the pyramidal cell layer of the CA1, CA2 and CA3 subfields, with the highest expression in the CA2 subfield, and in the granular cell layer of the dentate gyrus. Angiotensin II binding was detected in the stratum oriens and stratum radiatum of the CA1 and CA2 subfields, in the stratum oriens of the CA3 subfield, and in the molecular layer of the dentate gyrus. We then studied the effect of ischemia on hippocampal angiotensin II receptor expression, 1, 4 and 15 days after bilateral occlusion of the common carotid arteries for 5 min. No changes in angiotensin II receptor mRNA or binding were detected 1 day after ischemia. Delayed, progressive loss of angiotensin II mRNA and binding occurred 4 and 15 days after ischemia, in the CA1, CA2 and CA3 subfields. The decline was faster in the CA1 subfield, and paralleled the loss of neurons after ischemia. In the dentate gyrus, angiotensin II receptor mRNA and angiotensin II binding were not changed when compared to sham operated controls. The decrease of angiotensin II receptor expression may reflect the loss of angiotensin II receptor-producing neurons rather than a down-regulation of receptor expression.     

Effects of polyglycolic acid and polypropylene meshes on postoperative adhesion formation in mice

The purpose of this study was to investigate the effects of polyglycolic acid (PGA), an absorbable (ABS) mesh, and polypropylene (PP), a nonabsorbable (NA) mesh, on intestinal adhesion formation. Altogether 72 mice were divided into a control group of 24, an ABS mesh group of 23, and an NA mesh group of 25. All three groups were divided into two subgroups for evaluation of adhesion severity at postoperative (po) days 5 and 90. Adhesion severity was measured with adhesion grading and tissue hydroxyproline (OHP) levels. Adhesion degree was minimal (1) in all subjects on day 5. Also there was no difference in tissue OHP levels between three groups on day 5 (p > 0.05). Adhesion degree and tissue OHP levels as determinants of adhesion severity were higher in the PGA mesh group than the control group and the PP mesh group on day 90 (p < 0.001). There was no difference between the control group and the PP mesh groups (p > 0.05). Adhesion degree was higher on day 90 than on day 5 in the control group and the PGA mesh group (p < 0.05), whereas tissue OHP level was higher on day 90 than on day 5 in all three groups (p < 0.001). Also there was linear correlation between adhesion degree and tissue OHP levels (r = 0.86, p < 0.001). The study demonstrates that ABS PGA mesh has higher potential for adhesion formation than the NA PP mesh, probably related to the increased foreign body and inflammatory reactions during the absorption process of the mesh.     

Human anticardiolipin monoclonal autoantibodies cause placental necrosis and fetal loss in BALB/c mice

OBJECTIVE: To analyze the structure, specificity, and in vivo pathogenetic potential of 2 human anticardiolipin (aCL) monoclonal antibodies (MAb). METHODS: Human aCL IgG MAb were generated from hybridized Epstein-Barr virus-induced B cell lines from a healthy subject (MAb 519) and from a patient with primary antiphospholipid syndrome (MAb 516). Studies of antigen-binding specificity and analysis of Ig V-gene mutations were carried out. The MAb were independently injected into mated female BALB/c mice, and their effect on pregnancy outcome was compared with that of MAb 57, a highly mutated and antigen-selected human IgG1lambda rabies virus antibody. RESULTS: Both MAb 519 and MAb 516 utilized minimally mutated V(H)DJ(H) and VkappaJkappa gene segments and bound cardiolipin and other anionic phospholipids in the absence of beta2-glycoprotein I (beta2-GPI). The mice injected with aCL MAb displayed a significantly higher rate of fetal resorption and a significant reduction in fetal and placental weight as compared with those injected with MAb 57. These findings were accompanied by a finding of placental human IgG deposition and necrosis in the aCL MAb-treated animals. CONCLUSION: The results of this study indicate that human aCL IgG that are beta2-GPI independent can induce pathology.     

TNF-alpha messenger RNA and protein expression in the uteroplacental unit of mice with pregnancy loss

An elevated expression of TNF-alpha in embryonic microenvironment was found to be associated with postimplantation loss. In this work, we examined the pattern of TNF-alpha expression at both the mRNA and the protein level as well as the distribution of TNF-alpha receptor mRNA in the uteroplacental unit of mice with induced (cyclophosphamide-treated) or spontaneous (CBA/J x DBA/2J mouse combination) pregnancy loss. RNase protection analysis demonstrated an increase in TNF-alpha mRNA expression in the placentae of mice with pregnancy loss compared with that in control mice. TNF-alpha messages were localized to the uterine epithelium and stroma as well as the giant and spongiotrophoblast cells of the placenta. The intensity of the hybridization signal in placentae of mice with pregnancy loss was substantially higher than that in control mice. The up-regulation of TNF-alpha mRNA was accompanied by an increase in the expression of TNF-alpha receptor I mRNA in the same cell populations. The elevation of TNF-alpha production was also demonstrated at the protein level. Western blot analysis showed an increased level of the 18- and 26-kDa TNF-alpha protein species in the uteroplacental unit of mice with pregnancy loss. Immunostaining revealed TNF-alpha-positive leukocytes located in the uterus and placenta. Finally, we found that immunization of mice with cyclophosphamide-induced pregnancy loss while decreasing the resorption rate in these females resulted in a decline in TNF-alpha expression at the fetomaternal interface. These data clearly suggest an involvement of TNF-alpha in pathways leading to both spontaneous and induced placental death.     

Affinity maturation in Lyn kinase-deficient mice with defective germinal center formation

Lyn kinase-deficient (lyn-/-) mice show several abnormalities such as reduced numbers of circulating B cells, hyper-IgM, and low proliferative responses induced by CD40 ligand. Lyn-/- mice also develop splenomegaly, produce autoreactive Abs with age, and finally develop glomerulonephritis. Another abnormality observed in lyn-/- mice is that their disability to form germinal centers (GCs). It has been considered that GCs play an important role in affinity maturation and differentiation to B cell memory upon immunization with thymus-dependent Ag. Since Lyn kinase has been thought to be downstream of the signals from the B cell Ag receptor as well as CD40, we studied whether or not lyn-/- mice could exhibit normal Ag-specific class switching and affinity maturation following somatic hypermutation. The mice were immunized with (4-hydroxy-3-nitrophenyl)acetyl-chicken gamma-globulin (NP-CG). Production of NP-specific IgG1 Abs was slightly reduced but clearly detectable. The affinity of Abs produced was comparable to that in wild-type mice. Furthermore, somatic hypermutation occurred in the heavy-chain variable region at the same level as that in wild-type mice. Therefore, we conclude that isotype switching and affinity maturation occur normally in lyn-/- mice without the formation of GCs. The results lead to a speculation that Lyn may not play a role in induction of isotype switching or affinity maturation, despite being downstream of the signals from the B cell Ag receptor complex and CD40, and that GC architecture may not be absolutely essential for affinity maturation.