Characteristics of polyclonal anti-human nephrin antibodies induced by genetic immunization using nephrin cDNA.

BACKGROUND: Nephrin is an essential protein for maintaining the normal structure of the podocyte foot process and the glomerular filtration barrier. To analyse the mechanism of proteinuria and treatment of nephrotic syndrome, we produced and characterized polyclonal anti-human nephrin antibodies using a newly developed genetic immunization technique. METHODS: An expression vector with full-length or fragmented cDNA of human nephrin protein was administered to female Lewis rats once a week for 12 weeks using a gene-gun method. Antibody production against different fragments of nephrin protein was then analysed by ELISA, Western blot analysis, immunoprecipitation and FACS analysis. We also analysed recognition of native nephrin protein using immunohistochemistry and electron microscopy, and conducted a functional analysis of in vitro clustering of nephrin protein. RESULTS: Serum anti-nephrin antibody titers reached a maximum level at 8 or 12 weeks. Four of five antibodies induced with cDNA of five different Ig-like motif fragments showed antigen-specific binding to Escherichia coli and produced recombinant human nephrin protein. Only two of these four antibodies, plus one antibody induced by full-length human nephrin protein cDNA, bound to solubilized native nephrin protein. These three IgG antibodies, subclasses IgG1, IgG2a and IgG2b, showed fine granular staining along the glomerular basement membrane of normal human glomeruli and clustering of human nephrin protein on plasma membranes of HEK293 cells. CONCLUSIONS: We successfully produced polyclonal anti-human nephrin antibodies by genetic immunization using nephrin cDNA. These new antigen-specific polyclonal antibodies will be useful for functional analysis and tissue staining of native nephrin protein.     

Use of desferrioxamine in the treatment of aceruloplasminemia

Aceruloplasminemia is a newly recognized autosomal recessive disorder of iron metabolism resulting in neurodegeneration of the retina and basal ganglia. We report here on the treatment of a patient who developed progressive extrapyramidal symptoms that included blepharospasm, grimacing, and rigidity associated with increased iron deposition in the brain and visceral organs. Treatment for 10 months with the iron chelator desferrioxamine decreased brain iron stores, prevented progression of the neurological symptoms, and reduced plasma lipid peroxidation. These data suggest that early treatment with this chelator may be useful in such patients to diminish central nervous system iron accumulation and to prevent or ameliorate neurological symptoms associated with neurodegeneration.     

Comparison study of single and concurrent administrations of carbapenem, new quinolone, and macrolide against in vitro nontypeable Haemophilus influenzae mature biofilms

Nontypeable Haemophilus influenzae (NTHi) is an opportunistic pathogen and a common cause of otitis media in children, chronic bronchitis, and pneumonia in patients with chronic obstructive pulmonary disease. Many studies have reported that NTHi is capable of producing biofilms, which may be one of the important factors involved in chronic diseases and accelerating antimicrobial resistance. Unfortunately, there is still no consensus about the elimination of biofilms. In this study, concurrent administrations of levofloxacin (LVFX)-imipenem (IPM) and clarithromycin (CAM)-IPM, as well as the single administration of IPM, LVFX, and CAM, were performed to treat the mature biofilms produced by NTHi, respectively. Biofilm inhibition was quantified using microtiter biofilm assay (MBA), and relative biomass was calculated as the ratio compared to that of untreated control biofilms. The relative biomasses of biofilms treated with IPM, LVFX-IPM, and CAM-IPM against a β-lactamase-negative ampicillin-resistant strain was 1.10, 0.08, and 0.13 at 1× minimum inhibitory concentration (MIC), 0.90, 0.05, and 0.07 at 10× MIC, and 0.80, 0.06, and 0.07 at 100× MIC, respectively. Biofilms were also visually observed by scanning electron microscopy, and a focused ion-beam system showed that high concentrations of combined administration strongly inhibited the biofilms, which was consistent with the results of MBA. Our data demonstrated the antibiofilm effect of concurrent administration against mature NTHi biofilms, which indicated a rationale for the potential use of concurrent administrations in diseases involving chronic NTHi biofilms.     

Protein kinase C delta contributes to increase in EP3 agonist-induced contraction in mesenteric arteries from type 2 diabetic Goto-Kakizaki rats

Prostaglandin E₂ (PGE₂), an important and ubiquitously present vasoactive eicosanoid, may either constrict or dilate systemic vascular beds. However, little is known about the vascular contractile responsiveness to and signaling pathways for PGE₂ at the chronic stage of type 2 diabetes. We hypothesized that PGE₂-induced arterial contraction is augmented in type 2 diabetic Goto-Kakizaki (GK) rats via the protein kinase Cδ (PKCδ) pathway. Here, we investigated the vasoconstrictor effects of PGE₂ and of sulprostone (EP1-/EP3-receptor agonist) in rings cut from superior mesenteric arteries isolated from GK rats (37–44 weeks old). In arteries from GK rats (vs. those from age-matched Wistar rats), examined in the presence of a nitric oxide synthase inhibitor: 1) the PGE₂- and sulprostone-induced vasocontractions (which were not blocked by the selective EP1 receptor antagonist sc19220) were enhanced, and these enhancements were suppressed by rottlerin (selective PKCδ inhibitor) but not by Gö6976 (selective PKCα/β inhibitor); 2) the sulprostone-stimulated phosphorylation of PKCδ (at Thr⁵⁰⁵), which yields an active form, was increased and 3) sulprostone-stimulated caldesmon phosphorylations, which are related to isometric force generation in smooth muscle, were increased. The protein expression of EP3 receptor in superior mesenteric arteries was similar between the two groups of rats. Our data suggest that the diabetes-related enhancement of EP3 receptor-mediated vasocontraction results from activation of the PKCδ pathway. Alterations in EP3 receptor-mediated vasocontraction may be important factors in the pathophysiological influences over arterial tone that are present in diabetic states.     

Validation of the (Q)SAR combination approach for mutagenicity prediction of flavor chemicals

Most exposure levels of flavor in food are considered to be extremely low. If at all, genotoxic properties should be taken into account in safety evaluations. We have recently established a (quantitative) structure–activity relationship, (Q)SAR, combination system, which is composed of three individual models of mutagenicity prediction for industrial chemicals. A decision on mutagenicity is defined as the combination of predictive results from the three models. To validate the utility of our (Q)SAR system for flavor evaluation, we assessed 367 flavor chemicals that had been evaluated mainly by JECFA and for which Ames test results were available. When two or more models gave a positive evaluation, the sensitivity was low (19.4%). In contrast, when one or more models gave a positive evaluation, the sensitivity increased to 47.2%. The contribution of this increased sensitivity was mainly due to the result of the prediction by Derek for Windows, which is a knowledge-based model. Structural analysis of false negatives indicated some common sub-structures. The approach of improving sub-structural alerts could effectively contribute to increasing the predictability of the mutagenicity of flavors, because many flavors possess categorically similar functional sub-structures or are composed of a series of derivatives.     

Phosphorylation of ephrin-B1 regulates dissemination of gastric scirrhous carcinoma

Interaction of the Eph family of receptor protein tyrosine kinase and its ligand ephrin family induces bidirectional signaling via cell-cell contacts. High expression of B-type ephrin is frequently found in various cancer cells, and their expression levels are associated with high invasion of tumors and poor prognosis. However, whether ephrin-B1 actually promotes invasion of cancer cells in vivo has not been shown. We investigated the involvement of ephrin-B1 in regulating the invasiveness of scirrhous gastric cancer, which is a diffusely infiltrative carcinoma with high invasion potential. Reduction of ephrin-B1 expression by short inter-fering RNA or overexpression of phosphorylation-defective mutant suppressed migration and invasion of scirrhous gastric cancer cells in vitro without affecting tumor cell proliferation and apoptosis. Blocking of tyrosine phosphorylation of ephrin-B1 attenuates not only dissemination of cancer cells injected intraperitoneally but also local invasion and dissemination of orthotopically implanted cancer cells in the gastric wall of nude mice. Furthermore, blocking of ephrin-B1 phosphorylation attenuated the activation of Rac1 GTPase in these invasive gastric cancer cells. Our results suggest that tyrosine phosphorylation of ephrin-B1 promotes invasion of cancer cells in vivo and is a potential therapeutic target in some types of gastrointestinal cancers.     

Isolation, characterization, and mapping of Tn5 insertions into the 140-megadalton invasion plasmid defective in the mouse Sereny test in Shigella flexneri 2a

Using Shigella flexneri 2a YSH6000, we isolated 304 independent Tn5 insertion mutants in the 230-kilobase invasion plasmid, pMYSH6000. The site of each Tn5 insertion was assigned to 23 SalI fragments on the previously made SalI cleavage map of pMYSH6000. Among the 304 insertions, 150 were negative in expression of four phenotypes examined (mouse Sereny test [Ser], invasion into epithelial cells [Inv], Congo red binding [Pcr], and inhibition of bacterial growth [Igr] ): 12 were Ser- Inv+ Pcr+ Igr+, and 142 were positive in all four phenotypes. Tn5 insertions in the avirulent mutants were distributed in two separate SalI fragments, F and G, and in four contiguous SalI fragments, B, P, H, and D. Fragment G contains a novel class of determinant(s) which is required only for Ser+ but not for Inv+, Pcr+, and Igr+. Fragment F contains the previously characterized virF locus. B, P, H, and D each contained both virulent and avirulent Tn5 insertions. This indicates that more than two gene clusters exist within this region. Both are required for expression of all four virulence phenotypes.     

Expression of the p63 and Notch signaling systems in rat testes during postnatal development: comparison with their expression levels in the epididymis and vas deferens

The role of tubular structures that contribute to the passage of spermatozoa is not solely passive; these structures actively contribute to their own functions, although these tubules and ducts are contiguous and collaborate in the development of the male gamete along their lengths. The testis has the specific function to generate spermatozoa and spermatozoa undergo numerous changes as they pass through the epididymis. A member of the p53 family of genes, p63, is highly expressed in the basal layers of epithelial tissues and plays a key role in maintaining their cell populations, whereas Notch 1 and its ligand Jagged 2 have an important role in the differentiation of germ cells and Jagged 2 is up-regulated by TAp63, one of the p63 isoforms, which transactivates p53 target genes and induces apoptosis. Although the presence of p63 in most epithelia is established, the role of p63 and its possible relationship with the Notch system in the seminiferous epithelium have not been examined. Therefore, we investigated the expression of p63, Jagged 2, and Notch 1 in the testis during postnatal development in comparison with their expression levels in the vaso-epididymal epithelium. In the testis, the expression of TAp63 mRNA increased at day 14 after birth and the expressions of Jagged 2 and Notch 1 mRNA increased at day 16 after birth, suggesting that TAp63-mediated Jagged 2 induction activates the Notch signaling system. On the other hand, the strong signal of DeltaNp63 mRNA was already recognized in the vas deferens at day 0 after birth and advanced chronologically along the duct to the caput epididymis and p63 protein was expressed in basal cells in their epithelium, whereas the mRNAs of Jagged 2 and Notch 1 were maintained at a low level. Consequently, examination of our data raises the probability that TAp63 has an important role for maintenance of germ cell numbers, triggering or balancing the development, differentiation, and apoptosis of germ cells in the testis, which is completely different from the role of DeltaNp63 in other epithelial tissues.     

Upper cervical spine movement during intubation: fluoroscopic comparison of the AirWay Scope, McCoy laryngoscope, and Macintosh laryngoscope

BACKGROUND: The AirWay Scope (AWS) is a new fibreoptic intubation device, which allows visualization of the glottic structures without alignment of the oral, pharyngeal, and tracheal axes, and thus may be useful in patients with limited cervical spine (C-spine) movement. We fluoroscopically evaluated upper C-spine movement during intubation with the AWS or Macintosh or McCoy laryngoscope. METHODS: Forty-five patients, with normal C-spine, scheduled for elective surgery were randomly assigned to one of the three intubation devices. Movement of the upper C-spine was examined by measuring angles formed by adjacent vertebrae during intubation. Time to intubation was also recorded. RESULTS: Median cumulative upper C-spine movement was 22.3 degrees, 32.3 degrees, and 36.5 degrees with the AWS, Macintosh laryngoscope, and McCoy laryngoscope, respectively (P<0.001, AWS vs, Macintosh and McCoy). The AWS reduced maximum movement of the C-spine at C1/C2 in comparison with the Macintosh or McCoy laryngoscope (P=0.012), and at C3/C4 in comparison with the McCoy laryngoscope (P=0.019). Intubation time was significantly longer in the AWS group than in the Macintosh group (P=0.03). CONCLUSIONS: Compared with the Macintosh or McCoy laryngoscope, the AWS produced less movement of upper C-spine for intubation in patients with a normal C-spine.