A novel cadherin cell adhesion molecule: its expression patterns associated with implantation and organogenesis of mouse embryos.

The Ca2+-dependent cell adhesion molecules, termed cadherins, were previously divided into two subclasses, E- and N-types, with different adhesive specificity. In this study, we identified a novel class of cadherin, termed P-cadherin, using a visceral endoderm cell line PSA5-E. This cadherin was a 118,000-D glycoprotein and distinct from E- and N-cadherins in immunological specificity and molecular mass. In accord with these findings, cells with P-cadherin did not cross-adhere with cells with E-cadherin. P-Cadherin first appeared in developing mouse embryos in the extraembryonic ectoderm and the visceral endoderm at the egg cylinder stage and later was expressed in various tissues. The placenta and the uterine decidua most abundantly expressed this cadherin. The expression of P-cadherin was transient in many tissues, and its permanent expression was limited to certain tissues such as the epidermis, the mesothelium, and the corneal endothelium. When the tissue distribution of P-cadherin was compared with that of E-cadherin, we found that: each cadherin displayed a unique spatio-temporal pattern of expression; P-cadherin was co-expressed with E-cadherin in local regions of various tissues; and onset or termination of expression of P-cadherin was closely associated with connection or segregation of cell layers, as found with other cadherins. These results suggested that differential expression of multiple classes of cadherins play a role in implantation and morphogenesis of embryos by providing cells with heterogenous adhesive specificity.

Cloning and expression of cDNA encoding a neural calcium-dependent cell adhesion molecule: its identity in the cadherin gene family.

The neural cadherin (N-cadherin) is a Ca2+-dependent cell-cell adhesion molecule detected in neural tissues as well as in non-neural tissues. We report here the nucleotide sequence of the chicken N-cadherin cDNA and the deduced amino acid sequence. The sequence data suggest that N-cadherin has one transmembrane domain which divides the molecule into an extracellular and a cytoplasmic domain; the extracellular domain contains internal repeats of characteristic sequences. When the N-cadherin cDNA connected with virus promoters was transfected into L cells which have no endogenous N-cadherin, the transformants acquired the N-cadherin-mediated aggregating property, indicating that the cloned cDNA contained all information necessary for the cell-cell binding action of this molecule. We then compared the primary structure of N-cadherin with that of other molecules defined as cadherin subclasses. The results showed that these molecules contain common amino acid sequences throughout their entire length, which confirms our hypothesis that cadherins make a gene family.

Calcium-dependent cell-cell adhesion molecules (cadherins): subclass specificities and possible involvement of actin bundles.

Cadherins are a family of cell-cell adhesion molecules and are divided into subclasses with distinct adhesive specificities and tissue distribution. Here we examined the distribution of cadherins at contact sites between cells expressing the same or different cadherin subclasses. Each cadherin was concentrated at the boundary between cells expressing an identical cadherin subclass, irrespective of the cell types connected. However, such localization decreased or disappeared at the boundary between cells containing different cadherin subclasses. We also found that the localization of cadherins precisely coincided with that of actin bundles; both were detected at the apical region of cell sheets. This co-localization was retained even after cells were either treated with cytochalasin D or extracted with the detergent NP40. These results suggest that each cadherin subclass preferentially interacts with its own molecular type at intercellular boundaries, and that cadherin molecules may be associated with actin-based cytoskeletal elements.

Drosophila synapse formation: regulation by transmembrane protein with Leu-rich repeats, CAPRICIOUS.

Upon reaching the target region, neuronal growth cones transiently search through potential targets and form synaptic connections with only a subset of these. The capricious (caps) gene may regulate these processes in Drosophila. caps encodes a transmembrane protein with leucine-rich repeats (LRRs). During the formation of neuromuscular synapses, caps is expressed in a small number of synaptic partners, including muscle 12 and the motorneurons that innervate it. Loss-of-function and ectopic expression of caps alter the target specificity of muscle 12 motorneurons, indicating a role for caps in selective synapse formation.

Neural cadherin: role in selective cell-cell adhesion.

Cadherins are a family of Ca2+-dependent intercellular adhesion molecules. Complementary DNAs encoding mouse neural cadherin (N-cadherin) were cloned, and the cell binding specificity of this molecule was examined. Mouse N-cadherin shows 92 percent similarity in amino acid sequence to the chicken homolog, while it shows 49 percent and 43 percent similarity to epithelial cadherin and to placental cadherin of the same species, respectively. In cell binding assays, mouse N-cadherin did not cross-react with other mouse cadherins, but it did cross-react with chicken N-cadherin. The results indicate that each cadherin type confers distinct adhesive specificities on different cells, and also that the specificity of N-cadherin is conserved between mammalian and avian cells.

Ectopic expression of connectin reveals a repulsive function during growth cone guidance and synapse formation.

Connectin, a cell surface protein of the leucine-rich repeat family in Drosophila, is expressed on the surface of a subset of embryonic muscles (primarily lateral muscles), on the growth cones and axons of the motoneurons that innervate these muscles (primarily SNa motoneurons), and on several associated glial cells. When coupled with its ability to mediate homophilic cell adhesion, these results led to the suggestion that Connectin functions as an attractive signal for SNa pathfinding and targeting. In the present study, we ectopically expressed Connectin on ventral muscles normally innervated by SNb motoneurons. The SNb growth cones change both their morphology and their trajectory when they encounter ectopic Connectin-positive ventral muscles, displaying "bypass," "detour," and "stall" phenotypes. Moreover, SNb synapse formation is prevented by Connectin expression on ventral muscles. These results reveal a repulsive function for Connectin during motoneuron growth cone guidance and synapse formation.

Innervation and activity dependent dynamics of postsynaptic oxidative metabolism.

Despite extensive investigations into the mechanisms of aerobic respiration in mitochondria, the spontaneous metabolic activity of individual cells within a whole animal has not been observed in real time. Consequently, little is known about whether and how the level of mitochondrial energy metabolism is regulated in a cell during development of intact systems. Here we studied the dynamics of postsynaptic oxidative metabolism by monitoring the redox state of mitochondrial flavoproteins, an established indicator of energy metabolism, at the developing Drosophila neuromuscular junction. We detected transient and spatially synchronized flavoprotein autofluorescence signals in postsynaptic muscle cells. These signals were dependent on the energy substrates and coupled to changes in mitochondrial membrane potential and Ca2+ concentration. Notably, the rate of autofluorescence signals increased during synapse formation through contact with the motoneuronal axon. This rate was also influenced by the magnitude of synaptic inputs. Thus, presynaptic cells tightly regulate postsynaptic energy metabolism presumably to maintain an energetic balance during neuromuscular synaptogenesis. Our results suggest that flavoprotein autofluorescence imaging should allow us to begin assessing the progress of synapse formation from a metabolic perspective.

Synaptic components necessary for retrograde signaling triggered by calcium/calmodulin-dependent protein kinase II during synaptogenesis.

The development and function of presynaptic terminals are tightly controlled by retrograde factors presented from postsynaptic cells. However, it remains elusive whether major constituents of synapses themselves are necessary for retrograde modulation during synaptogenesis. Here we show that the homophilic cell adhesion molecule Fasciclin II (FasII) as well as the scaffolding protein Discs large (DLG) is indispensable for retrograde signaling initiated by calcium/calmodulin-dependent protein kinase II (CaMKII) at developing Drosophila neuromuscular junctions. Postsynaptic activation of CaMKII increased the area of nerve terminals, the number of active zones, and the frequency of miniature excitatory synaptic currents in wild-type animals. However, all of these retrograde effects were abolished in the fasII or dlg mutant background. On the other hand, the retrograde effects remained in null mutants of the glutamate receptor subunit GluRIIA. Furthermore, we show that CaMKII-induced modulation was independent of the bone morphogenetic protein signaling that is important for retrograde control at mature larvae. These results highlight a novel function of FasII as well as DLG, and more broadly, illustrate that prime synaptic components are necessary for transferring target-derived signals to presynaptic cells at a certain developing synapse.

Effect of Bifidobacterium bifidum fermented milk on Helicobacter pylori and serum pepsinogen levels in humans.

Helicobacter pylori infection is an important risk factor for gastric diseases. Some probiotics are useful for suppressing H. pylori infection. Bifidobacterium bifidum YIT 4007 can improve the experimental gastric injury in rats and the disease stages on the gastric mucosa in peptic ulcer patients. We evaluated the fermented milk using a clone (BF-1) having the stronger ability to survive in the product than this parent strain to clarify the in vitro suppressive effect of BF-1 on H. pylori and the in vivo efficacy of BF-1 fermented milk on H. pylori and gastric health. In the mixed culture assay of BF-1 and H. pylori, the number of pathogens was decreased such that it was not detected after 48 h in the Brucella broth with a decrease in pH values. In the cell culture experiment with human gastric cells, the H. pylori infection-induced IL-8 secretion was suppressed by the preincubation of BF-1. In a human study of 12-wk ingestion (BF-1 group, n = 40; placebo group, n = 39) with a randomized double-blind placebo-control design, the H. pylori urease activity and gastric situation were evaluated using a urea breath test (UBT) and the serum pepsinogen (PG) levels as biomarkers for inflammation or atrophy, respectively. In the H. pylori-positive subjects, the difference (DeltaUBT) of the UBT value from the baseline value in the BF-1 group (n = 34) was lower than that in the placebo group (n = 35) at 8 wk. The baseline UBT values showed a negative correlation with DeltaUBT values at 8 and 12 wk in the BF-1 group but not in the placebo. In the PG-positive subjects classified by the PG test method, the BF-1 group was lower in DeltaUBT values than the placebo group at 8 and 12 wk. In the active gastritis class by PG levels, the BF-1 group was lower in their DeltaUBT values than the placebo at 8 and 12 wk. The PG I levels in the BF-1 group were lower than the placebo at 12 wk. The PG II levels in the BF-1 group did not change during the ingestion period, but the placebo was increased. The PG I/II ratios slightly decreased from baseline at 12 and 20 wk in the BF-1 and placebo groups. These patterns were also observed in the H. pylori-positive subjects. The improving rates of upper gastrointestinal symptomatic subjects and total symptom numbers in the BF-1 group were higher than those in the placebo. These results indicate that BF-1 fermented milk may affect H. pylori infection or its activity, gastric mucosal situation, and the emergence of upper gastrointestinal symptoms.

Angiotensin AT(1) and AT(2) receptors differentially regulate angiopoietin-2 and vascular endothelial growth factor expression and angiogenesis by modulating heparin binding-epidermal growth factor (EGF)-mediated EGF receptor transactivation.

Angiotensin II (Ang II)-mediated signals are transmitted via heparin binding epidermal growth factor (EGF)-like growth factor (HB-EGF) release followed by transactivation of EGF receptor (EGFR). Although Ang II and HB-EGF induce angiogenesis, their link to the angiopoietin (Ang)-Tie2 system remains undefined. We tested the effects of Ang II on Ang1, Ang2, or Tie2 expression in cardiac microvascular endothelial cells expressing the Ang II receptors AT(1) and AT(2). Ang II significantly induced Ang2 mRNA accumulations without affecting Ang1 or Tie2 expression, which was inhibited by protein kinase C inhibitors and by intracellular Ca(2+) chelating agents. Ang II transactivated EGFR via AT(1), and inhibition of EGFR abolished the induction of Ang2. Ang II caused processing of pro-HB-EGF in a metalloproteinase-dependent manner to stimulate maturation and release of HB-EGF. Neutralizing anti-HB-EGF antibody blocked EGFR phosphorylation by Ang II. Ang II also upregulated vascular endothelial growth factor (VEGF) expression in an HB-EGF/EGFR-dependent manner. AT(2) inhibited AT(1)-mediated Ang2 expression and phosphorylation of EGFR. In an in vivo corneal assay, AT(1) induced angiogenesis in an HB-EGF-dependent manner and enhanced the angiogenic activity of VEGF. Although neither Ang2 nor Ang1 alone induced angiogenesis, soluble Tie2-Fc that binds to angiopoietins attenuated AT(1)-mediated angiogenesis. These findings suggested that (1) Ang II induces Ang2 and VEGF expression without affecting Ang1 or Tie2 and (2) AT(1) stimulates processing of pro-HB-EGF by metalloproteinases, and the released HB-EGF transactivates EGFR to induce angiogenesis via the combined effect of Ang2 and VEGF, whereas AT(2) attenuates them by blocking EGFR phosphorylation. Thus, Ang II is involved in the VEGF-Ang-Tie2 system via HB-EGF-mediated EGFR transactivation, and this link should be considerable in pathological conditions in which collateral blood flow is required.

Cytoplasmic antineutrophil cytoplasmic antibody positive pauci-immune glomerulonephritis associated with infectious endocarditis.

Renal deterioration often occurs in cases of infectious endocarditis (IE), but, IE- associated nephritis with rapidly progressive glomerulonephritis (RPGN) is rare. Patients with severe infection (e.g., IE) sometimes show positivity for cytoplasmic antineutrophil cytoplasmic antibodies (C-ANCA). Therefore, diagnosis and treatment are very difficult in cases of RPGN with IE and positivity for C-ANCA. Such cases are rare, only 12 have been reported in the English literature. Herein, we describe the case of a 50-year-old man who presented with RPGN with IE and tested positively for C-ANCA. He was referred to our hospital because of leg edema, purpura and renal dysfunction. Laboratory tests revealed serum creatinine elevation and positivity for C-ANCA and proteinase 3-specific (PR3)-ANCA. RPGN and acute renal failure were diagnosed. Hemodialysis and steroid therapy were started. Streptococcus oralis was isolated by blood culture. Transthoracic echocardiography revealed grade III mitral valve insufficiency with two vegetations. Therefore, IE was diagnosed. The steroid therapy was stopped, and antibiotic therapy was begun. Because there was no improvement, surgical therapy was performed. The operation was successful, but the patient died of brain hemorrhage. Our experience in this case indicates C/PR3-ANCA positive RPGN must be ruled out in patients with infectious disease, particularly IE, together with renal symptoms, and renal biopsy should be performed.

Postsynaptic activation of calcium/calmodulin-dependent protein kinase II promotes coordinated pre- and postsynaptic maturation of Drosophila neuromuscular junctions.

The interaction between a neuron and its target cell(s) is essential for the development of synapses. To elucidate the role of target cells in synaptogenesis, the activity of postsynaptic calcium/calmodulin-dependent protein kinase II (CaMKII) was manipulated in a mosaic manner and its specific effect was examined at the developing Drosophila neuromuscular junction. We found that postsynaptic expression of constitutively active CaMKII augmented the amplitude of excitatory synaptic currents (ESCs) and the frequency of miniature ESCs. It also promoted morphological maturation of presynaptic as well as postsynaptic specializations, which presumably underlie the enhancement of synaptic activities. Expression of an inhibitory peptide of CaMKII in the postsynaptic cell partially affected the synaptic maturation. These results suggest two significant functions of postsynaptic CaMKII in synaptogenesis-retrograde modulation of presynaptic properties and coordinated regulation of pre- and postsynaptic maturation.

Developmental stage-dependent modulation of synapses by postsynaptic expression of activated calcium/calmodulin-dependent protein kinase II.

In Drosophila neuromuscular junctions, there is a unique system which consists of two neighboring muscles (M6 and M7) innervated by the same neurons and a gene of interest can be expressed in only M6 or in both muscles by GAL4-upstream activating sequence expression system. By using this system, we previously demonstrated that expression of activated calcium/calmodulin-dependent protein kinase II (CaMKII) in the muscle cell promotes coordinated maturation of pre- and postsynaptic sites of larvae just after hatching (JAH larvae) in a synapse-specific manner. Here we show that the promotive effects are no longer seen in the older larvae, 8-10 h after hatching (8 h AH larvae). Morphological studies indicate that CaMKII activation in fact reduces postsynaptic sites at 8 h AH. This is opposite to the effect observed in JAH larvae. These results suggest that the mode of CaMKII function switches during development, and that regulation of postsynaptic CaMKII activity is necessary for normal synaptic development. Finally, we report that in 8 h AH but not in JAH larvae, synapses on M7, in which CaMKII activity is not manipulated, are affected by the expression of activated CaMKII in M6. This suggests the interesting possibility that at certain developmental stages only, modification of synapses on one target cell can influence the synapses on another target cell innervated by the same neurons.

Safety and availability of doxazosin in treating hypertensive patients with chronic renal failure.

Hypertension accelerates the progression of renal disease in patients with chronic renal failure. Doxazosin, an alpha1-antagonist, is an antihypertensive agent with a long half-life. In this study, 15 patients with chronic renal failure were treated only with doxazosin and diuretics for 6 months and their blood pressure, renal parameters and lipid profile were measured. The initial dose of doxazosin was 2 mg/day and it was titrated until blood pressure was normalized. The average dose was 5.6 mg/day. As expected, systolic and diastolic blood pressure were decreased with treatment (165/91 mmHg to 135/73 mmHg). The drop in blood pressure was associated with an increase in glomerular filtration and a decrease in plasma BUN and creatinine levels. Reduction in mean blood pressure and decrease in proteinuria had a significant positive correlation (r=0.048, p=0.007). Proteinuria was decreased from 1.8 mg/day to 1.3 mg/day with doxazosin treatment and triglycerides also decreased, while HDL-cholesterol was increased. No side effects were observed. These results indicate that doxazosin is an efficient depressor agent with renal protective actions and that higher doses of doxazosin can be safely given to patients with chronic renal failure.

Membranous glomerulonephritis associated with hepatitis C virus infection: case report and literature review.

We describe the case of a 51-year-old man with hepatitis C virus (HCV) infection and a 3-month history of facial edema. Laboratory tests upon admission for renal biopsy showed normal renal function and normocomplementemia. Serum HCV antibody (Ab) and cryoglobulin were positive. Renal biopsy specimens showed features of membranous glomerulonephritis. The likely cause was immune complex-mediated glomerulonephritis associated with HCV infection. Reports of similar cases in the literature show the normocomplementemia and negative or slightly positive cryoglobulins observed in our case as well as seropositivity for circulating immune complexes containing HCV RNA. In our case, electron microscopic examination of the subepithelial glomerular lesions revealed massive virus-like particles within unusual multilayers of electron-dense deposits (EDDs), suggesting the existence of HCV in the glomeruli. In the addition to the unique histopathological feature the presence of La/SS-B antibody in his serum indicated an abnormal immune response associated with HCV. We advise him to undergo the therapy with new type of IFN such as pegIFN-alpha2a and/or anti-viral agent like ribavirin to achieve clinical and histopathological improvement.

Renal blood flow measurement with contrast-enhanced harmonic ultrasonography: evaluation of dopamine-induced changes in renal cortical perfusion in humans.

BACKGROUND: An accessible non-invasive method for evaluating renal regional blood flow in real time is highly desirable in the clinical setting. Recent progress in ultrasonography with microbubble contrast has allowed quantification of regional blood flow in animal models. AIMS: Goal ofthis study was to establish a convenient contrast--enhanced harmonic ultrasonography (CEHU) method for evaluating renal cortical blood flow in humans. METHODS: We carried out intermittent second harmonic imaging in 9 healthy volunteers. Pulse interval was progressively decreased from 4 s - 0.2 s during continuous venous infusion of the microbubble contrast agent. RESULTS: Pulse interval versus CEHU-derived acoustic intensity plots provided microbubble velocity (MV) and fractional vascular volume (FVV) during renal cortical perfusion in humans. Low-dose dopamine infusion (2 microg/min/kg) resulted in a significant increase in MV which correlated well with the increase in total renal blood flow (RBF) determined by a conventional study of p-aminohippurate clearance (C(PAH)) (r = 0.956, p < 0.0001). Although FVV was not significantly increased, alterations in CEHU-derived renal cortical blood flow calculated by the products of MV and FVV were also correlated with alterations in total RBF (r = 0.969, p < 0.0001). Thus, low-dose dopamine infusion increases renal cortical blood flow observed in CEHU, mainly by increasing MV. CONCLUSIONS: The present study shows that renal cortical blood flow in humans can be measured non-invasively by CEHU and that CEHU can be used for quantitatively evaluating changes induced by a therapeutic agent such as dopamine in flow velocity and in FVV.

Association of parvovirus B19 infection with acute glomerulonephritis in healthy adults: case report and review of the literature.

An otherwise healthy 20-year-old woman presented with an erythematous rash on her face as well as arthralgia and anemia. She also had systemic edema, proteinuria and hypertension. Laboratory data on admission showed hypocomplementemia, human parvovirus B 19 (HPV) DNA and both immunoglobulin (Ig) M and IgG antibodies to HPV in her serum. Renal biopsy specimens showed features of endocapillary glomerulonephritis under light microscopy. Electron microscopy showed massive subendothelial electron-dense deposits. No cause was probable other than immune complex-mediated glomerulonephritis associated with HPV infection. In a review of this and similar cases reported in the literature, several characteristic features come to light: female dominance, onset in the second or third decade of life, hypocomplementemia, histologic renal endocapillary and/or mesangioproliferative glomerulonephritis with subendothelial deposits and spontaneous recovery.

Subunit-specific and homeostatic regulation of glutamate receptor localization by CaMKII in Drosophila neuromuscular junctions.

For the efficient transfer of information across neural circuits, the number of synaptic components at synapses must be appropriately regulated. Here, we found that postsynaptic calcium/calmodulin dependent protein kinase II (CaMKII) modulates the localization of glutamate receptors (GluRs) at Drosophila larval neuromuscular junctions (NMJs). Expression of an inhibitory peptide of CaMKII, Ala, in muscle cells enhanced the density of GluRIIA, which is a major and calcium-permeable subunit of GluR, at synapses of third instar larval NMJs. On the other hand, postsynaptic expression of a constitutively active form of CaMKII (T287D) reduced synaptic GluRIIA. These results suggest that CaMKII regulates GluRIIA at NMJs. Moreover, postsynaptic expression of T287D abolished the accumulation of the scaffolding protein discs large (DLG) at synapses, while exerting no significant effects on the presynaptic area and the localization of cell adhesion molecule fasciclin II (FasII). The amplitude of excitatory junctional potentials (EJPs) was enhanced in Ala-expressing larvae, whereas it was unaffected in T287D-expressing larvae in spite of the prominent loss of GluRIIA. The amplitude of miniature EJPs (mEJPs) was significantly reduced and quantal content was significantly increased in T287D-expressing larvae. Notably, another class of GluR containing GluRIIB was enhanced by the postsynaptic expression of T287D. These results suggest that the homeostatic mechanism in T287D larvae works to maintain the level of synaptic responses. Thus, the Drosophila larval NMJs have several regulatory systems to ensure efficient muscle excitability which is necessary for proper larval movement.