Mouse semaphorin H induces PC12 cell neurite outgrowth activating Ras-mitogen-activated protein kinase signaling pathway via Ca(2+) influx.

We recently showed that mouse semaphorin H (MSH), a secreted semaphorin molecule, acts as a chemorepulsive factor on sensory neurites. In this study, we found for the first time that MSH induces neurite outgrowth in PC12 cells in a dose-dependent manner. Comparison of Ras-mitogen-activated protein kinase (MAPK) signaling pathways between MSH and nerve growth factor (NGF) revealed that these pathways are crucial for MSH action as well as NGF. K-252a, an inhibitor of tyrosine autophosphorylation of tyrosine kinase receptors (Trks), did not inhibit the action of MSH, suggesting that MSH action occurs via a different receptor than NGF. L- and N-types of voltage-dependent Ca(2+) channel blockers, diltiazem and omega-conotoxin, inhibited MSH-induced neurite outgrowth and MAPK phosphorylation in a Ca(2+)-dependent manner. A transient elevation in intracellular Ca(2+) level was observed upon MSH stimulation. These findings suggest that extracellular Ca(2+) influx, followed by activation of the Ras-MAPK signaling pathway, is required for MSH induced PC12 cell neurite outgrowth.

Mouse semaphorin H inhibits neurite outgrowth from sensory neurons.

Mouse semaphorin H (M-semaH) was structurally similar to semaphorin III/D, a mammalian homologue of collapsin 1 which was identified as a collapsing factor for sensory nerves. In this study we investigated the expression patterns of M-semaH mRNA and the protein binding sites in the trunk of mouse embryos. M-semaH mRNA was expressed in the mesenchymal tissues surrounding each dorsal root ganglia. These tissues include the caudal sclerotome and perinotochordal mesenchyme, which were thought to express factors repulsive to axons. M-semaH binding was detected on the spinal nerves. We further investigated, using in vitro co-culture assay, whether M-semaH acted as a chemorepulsive molecule on sensory axons. The results suggested that M-semaH was a candidate for a chemorepellent expressed in the mesenchyme surrounding the sensory ganglia, which is involved in the axonal guidance mechanism of sensory nerves in the trunk.

Microbial contamination of 'sterile water' used in Japanese hospitals.

We examined the following samples of water from 10 hospitals for microbial contamination: water obtained using an ultra filtration system (UF water), a reverse osmosis system (RO water), a water distillation system (distilled water) and tap water. UF water and RO water are used for handwashing before surgery, and distilled water for the preparation of drugs. All 10 samples of tap water examined were contaminated with < 10 colony forming units (cfu)/mL. Thirteen (68%) of 19 samples of UF water, nine (53%) of 17 samples of RO water and 15 (79%) of 19 samples of distilled water were contaminated with 10(1)-10(4) cfu/mL. The majority of micro-organisms were non-fermentative bacteria such as Sphingomonas paucimobilis and CDC gr. IV C-2. Japanese hospitals commonly use UF water and RO water for preoperative handwashing under the assumption that it is sterile. Our results suggest, however, that these types of water are inferior microbiologically to tap water. Distilled water from the dispensary was also contaminated with micro-organisms. The available chlorine content of tap water was 0.17-0.42 ppm and that of UF water (from tap water) 0-0.06 ppm. There was no available chlorine in RO water or distilled water (each from tap water). The reduction or disappearance of available chlorine appears to be associated with microbial contamination of UF water, RO water and distilled water.

[Study on the mechanism of placental transport of phosphate (using human placental microvillous (brush border) membrane vesicles)].

Using the rapid filtration technique, the uptake of phosphate into microvillous (brush border) membrane vesicles isolated from human placental trophoblast was investigated. The microvillous membrane vesicles exhibit the uptake of phosphate into an osmotically reactive intravesicular space and preincubation with the phosphate increased the uptake of phosphate into the vesicles. These findings indicate that the uptake of phosphate by placental trophoblast microvillous membranes represents transport into membrane vesicles. In the presence of sodium electrochemical gradient (extravesicular greater than intravesicular) the uptake of phosphate by vesicles shows an overshoot phenomenon. Sodium-dependent phosphate uptake is about two times higher at pH 6.0 as in the uptake observed at pH 8.0. The initial rate of sodium-dependent phosphate transport exhibited saturation kinetics with respect to phosphate concentration: An apparent Km of 0.28 mM and Vmax of 330 pmol/mg protein in 20 seconds were calculated. These results indicate that the transport of phosphate across the microvillous membranes is carrier mediated. Experiments with different anions (SCN-, Cl-, gluconate-) and ionophore (valinomycin) showed that at pH 7.4 phosphate transport in the presence of a Na+ gradient is almost independent of electrical potential across the vesicle membranes. It was concluded that placental trophoblast microvillous membranes contain an electroneutral Na+/phosphate co-transport system.

[Nephritogenic glycopeptide (nephritogenoside): its existence in the urine of toxemia patients].

Previously we reported that we succeeded in isolating from human full term placenta, glycopeptide which was immunologically the same as nephritogenic glycopeptide (Nephritogenoside) prepared from human renal GBM (glomerular basement membrane). This time to clarify the correlation between the pathology of toxemia and Nephritogenoside, we examined the existence of Nephritogenoside in the urine of toxemia patients. To purify Nephritogenoside from the urine powder of toxemia patients, we use the same procedures (enzyme digestion, Con A affinity column chromatography e.t.c.) as used in purifying Nephritogenoside from human full term placental TrBM (trophoblast basement membrane). In the urine of toxemia patients we clarified the existence of glycopeptide, which had specific affinity with Con A and was immunologically the same as Nephritogenoside. The existence of Nephritogenoside in the urine of toxemia patients further strengthened the possibility of a relationship between Nephritogenoside and the pathology of toxemia.

[Study on the mechanism of placental transport of L-lysine (using human placental microvillous (brush border) membrane vesicles)].

The uptake of L-lysine in human placental microvilli vesicles prepared from human term placenta was studied using the rapid filtration technique. The uptake of L-lysine into the vesicles was osmotically sensitive. This finding indicates that the uptake of L-lysine by placental microvilli vesicles represents transport into the membrane vesicles. The uptake of L-lysine into microvilli vesicles is sodium independent. The initial rate of uptake is markedly increased when the intravesicular space is rendered electrically more negative by membrane diffusion potential induced by the use of highly permeant anions or by K+ diffusion potentials via valinomycin. These results indicate that the sodium independent uptake of L-lysine into the microvilli membrane vesicles is dependent on the electrical potential difference of the membranes. A kinetic analysis of the uptake demonstrated that two transport systems for vesicular entry of L-lysine existed with a Km1 of 0.13 mM, Vmax1 of 590 pmol/mg protein/20 sec, Km2 of 0.91 mM, Vmax2 of 2010 pmol/mg protein/20 sec. The uptake of L-lysine in microvilli vesicles was inhibited by dibasic amino acid (L-arginine, L-ornithine, L-glutamine), but not by other classes of amino acid. These results indicate the existence of a dibasic amino acid specific transport system in placental microvilli membrane.

[Studies on L-glutamate transport mechanism in human placental trophoblast microvilli membrane vesicles].

The uptake of L-glutamate in brush border (microvilli) vesicles prepared from human term placenta was studied using the rapid filtration technique. The uptake of L-glutamate into the vesicles occurred osmotically, and preincubation with L-glutamate increased the uptake of amino acid. These findings indicate that the uptake of L-glutamate by placental trophoblast brush border membranes represents the transport into membrane vesicles. A Na+ electrochemical gradient (extravesicular greater than intravesicular) stimulated the initial rate of L-glutamate uptake about three times. The initial rate of transport exhibited saturation kinetics with respect to the L-glutamate concentration; an apparent Km of 0.15 mM and V max of 70 pmol/mg protein in 20 seconds were calculated. The uptake of L-glutamate into the vesicles was competitively inhibited by L-glutamate and L-cysteate (acidic amino acid). These results indicate that a Na-dependent acidic amino acid specific transport system exists in the placental trophoblast microvilli membrane. These results indicate that the transport of L-glutamate across the placental microvilli membrane is sodium-dependent and carrier mediated.

[Nephritogenic glycoprotein isolated from human placenta--purification and isolation by sonication].

Previously, we reported that we succeeded in isolating from human placenta a substance comparable to human renal nephritogenic glycoprotein prepared from glomerular basement membrane (GBM). This time we purified a sample isolated from human placental trophoblast basement membrane (TrBM) with the methods for purifying renal nephritogenic glycoprotein and clarified its chemical composition. To purify the sample prepared from human placental TrBM, we fractionated the sample by Zone Electrophoresis, Concanavalin A(Con A) affinity column chromatography and Bio Gel P200 column chromatography. As the active fraction of the sample purified from human placental TrBM bound specifically with Con A, it proved to be a glycoprotein. The monosaccharide composition of this glycoprotein was rich in glucose (glucose, galactose and mannose were in the ratio of 1.00:0.30:0.33) and the amino acid composition of this glycoprotein contained no collagenous components. Ouchterlony gel diffusion demonstrated the existence of a common precipitin line between antiserum to human renal nephritogenic glycoprotein prepared from GBM and human renal nephritogenic glycoprotein prepared from GBM, purified human placental glycoprotein, the sample prepared from human placental TrBM by sonication and nephritogenic glycoprotein prepared from urine of S.L.E. patient.

[Placental transport of taurine in brush border microvilli].

Placental transport of taurine was studied in isolated brush border microvillous plasma membrane vesicles by a rapid filtration technique. Brush border microvillous plasma membrane vesicles were prepared from syncytio trophoblast of human term placenta by a method of differential centrifugation and calcium precipitation. The specific activities of alkaline phosphatase, 5' nucleotidase and gamma-GTP in the membrane preparation were enriched to 13-14 times, 12-13 times, and 5-6 times respectively, as high as those in the homogenate. The membrane vesicles exhibit uptake of 3H-labeled taurine into an osmotically reactive intravesicular space. Taurine uptake by vesicles was stimulated specifically by an inward sodium gradient, and replacement of NaCl in the transport medium by KCl, LiCl, and choline chloride had no effect on the transport activity of the vesicles. Taurine transport is inhibited competitively by the presence of beta alanine and GABA. The initial rate of transport followed saturation kinetics with respect to the taurine concentration: An apparent Km of 0.22mM and Vmax of 67 pmol/mg protein were calculated in 20 seconds. These results indicate that transport of taurine across the placental brush border membrane is sodium dependent and carrier mediated.