Citron, a Rho-target, interacts with PSD-95/SAP-90 at glutamatergic synapses in the thalamus.

Proteins of the membrane-associated guanylate kinase family play an important role in the anchoring and clustering of neurotransmitter receptors in the postsynaptic density (PSD) at many central synapses. However, relatively little is known about how these multifunctional scaffold proteins might provide a privileged site for activity- and cell type-dependent specification of the postsynaptic signaling machinery. Rho signaling pathway has classically been implicated in mechanisms of axonal outgrowth, dendrogenesis, and cell migration during neural development, but its contribution remains unclear at the synapses in the mature CNS. Here, we present evidence that Citron, a Rho-effector in the brain, is enriched in the PSD fraction and interacts with PSD-95/synapse-associated protein (SAP)-90 both in vivo and in vitro. Citron colocalization with PSD-95 occurred, not exclusively but certainly, at glutamatergic synapses in a limited set of neurons, such as the thalamic excitatory neurons; Citron expression, however, could not be detected in the principal neurons of the hippocampus and the cerebellum in the adult mouse brain. In a heterologous system, Citron was shown to form a heteromeric complex not only with PSD-95 but also with NMDA receptors. Thus, Citron-PSD-95/SAP-90 interaction may provide a region- and cell type-specific link between the Rho signaling cascade and the synaptic NMDA receptor complex.

Reduction of renal transforming growth factor-beta activity without aggravation of growth retardation in nephritic rats by a methionine-threonine-supplemented low-casein diet.

The effects of a low-casein diet fortified with methionine and threonine on renal cortical and glomerular transforming growth factor (TGF)-beta activity were studied in rats with nephritis induced by anti-rat kidney glomerular basement membrane antiserum. Both normal and nephritic rats were fed experimental diets for 10 days. An injection of nephrotoxic serum increased urinary protein excretion and renal TGF-beta activity. A methionine-threonine-supplemented 8.5% casein diet, compared with a basal 20% casein diet, decreased these two measurements without aggravating growth retardation in nephritic rats. These results suggest that aggravation and alleviation of symptoms incident to anti-GBM nephritis are relevant to elevation and reduction of TGF-beta activity, respectively. The results also suggest that amino acid-balanced low-protein diets would have beneficial effects on glomerulonephritis without causing severe protein malnutrition.

The small GTP-binding protein Rho binds to and activates a 160 kDa Ser/Thr protein kinase homologous to myotonic dystrophy kinase.

The small GTP-binding protein Rho functions as a molecular switch in the formation of focal adhesions and stress fibers, cytokinesis and transcriptional activation. The biochemical mechanism underlying these actions remains unknown. Using a ligand overlay assay, we purified a 160 kDa platelet protein that bound specifically to GTP-bound Rho. This protein, p160, underwent autophosphorylation at its serine and threonine residues and showed the kinase activity to exogenous substrates. Both activities were enhanced by the addition of GTP-bound Rho. A cDNA encoding p160 coded for a 1354 amino acid protein. This protein has a Ser/Thr kinase domain in its N-terminus, followed by a coiled-coil structure approximately 600 amino acids long, and a cysteine-rich zinc finger-like motif and a pleckstrin homology region in the C-terminus. The N-terminus region including a kinase domain and a part of coiled-coil structure showed strong homology to myotonic dystrophy kinase over 500 residues. When co-expressed with RhoA in COS cells, p160 was co-precipitated with the expressed Rho and its kinase activity was activated, indicating that p160 can associate physically and functionally with Rho both in vitro and in vivo.

Protein kinase N (PKN) and PKN-related protein rhophilin as targets of small GTPase Rho.

The Rho guanosine 5'-triphosphatase (GTPase) cycles between the active guanosine triphosphate (GTP)-bound form and the inactive guanosine diphosphate-bound form and regulates cell adhesion and cytokinesis, but how it exerts these actions is unknown. The yeast two-hybrid system was used to clone a complementary DNA for a protein (designated Rhophilin) that specifically bound to GTP-Rho. The Rho-binding domain of this protein has 40 percent identity with a putative regulatory domain of a protein kinase, PKN. PKN itself bound to GTP-Rho and was activated by this binding both in vitro and in vivo. This study indicates that a serine-threonine protein kinase is a Rho effector and presents an amino acid sequence motif for binding to GTP-Rho that may be shared by a family of Rho target proteins.

Use of standardized protease enzymes for antibody screening of blood donor samples with the microplate system AutoAnalyzer.

Papain, bromelin and ficin can be standardized by the casein method for use in red blood cell antibody screening tests. The minimal and optimal enzyme activity for detecting blood group antibodies in donors, using the new Automated Pre-Transfusion Blood Testing System Olympus PK7200 by the two-stage method for all three enzymes, is one casein unit. One casein unit of proteinase activity changed the red blood cell surface charge to a low plateau value as measured by electrophoretic mobility and sialic acid content, and removed sterically inhibiting structures of surface protein as detected by SDS-PAGE.

Reduction of hyperlipidemia and proteinuria without growth retardation in nephritic rats by a methionine-supplemented, low-soy-protein diet.

Effect of a low-soy-protein-isolate (SPI) diet supplemented with methionine on hyperlipidemia, proteinuria, and hypoalbuminemia was studied in rats with nephrotoxic serum nephritis (NSN). Rats were fed experimental diets for 14 d after an injection of nephrotoxic serum. An 8.5%-SPI diet (8.5S), as compared with a basal 20%-SPI diet (20S), improved the hyperlipidemia, proteinura, and hypoalbuminemia secondary to NSN but retarded the growth of rats. The addition of 0.3% methionine to 8.5S (8.5SM) alleviated the growth retardation without loss of the above-mentioned beneficial effects. 8.5SM was found to suppress hepatic cholesterol synthesis compared with 20S. These results suggest that the methionine-supplemented low-SPI diet has a beneficial effect on hyperlipidemia, proteinuria, and hypoalbuminemia without inducing either growth retardation or severe fatty liver in nephritis. They also suggest that the hypocholesterolemic effect of 8.5SM in nephritic rats may be partly attributable to reduced hepatic cholesterol synthesis.

Prediction of engraftment after peripheral blood stem cell transplantation by CD34-positive cells in grafts.

A total of 91 peripheral blood stem cell collections were performed in 26 children with various malignant tumors and peripheral blood stem cell transplantations (PBSCT) were performed in 15 of the children. There was a positive correlation between logarithm of total CD34+ cells/kg and logarithm of colony-forming unit-granulocyte macrophage (CFU-GM)/kg (r = 0.86). The time elapsed until the white blood cells (WBC) exceeded 1000/microL was related to both CFU-GM (r = 0.67) and CD34+ cell count (r = 0.60). The number of days elapsed until platelet count exceeded 5 x 10(4)/microL was not related to the logarithm of CFU-GM count/10(5) per kg transfused (r = 0.47), but was related to the logarithm of CD34+ cell counts/10(6) per kg transfused (r = 0.73). The number of days elapsed until the reticulocytes exceeded 10% was not related to the logarithm of CFU-GM count/10(5) per kg (r = 0.52), but was related to the logarithm of CD34+ cell counts/10(6) per kg transfused (r = 0.91). Although CD34+ cell counts correlated with the number of CFU-GM, bone marrow regeneration rates in three lineages were predicted more accurately by the number of CD34+ cells transfused than by the number of CFU-GM. These results suggest that measurement of the CD34+ cell count may be useful in predicting bone marrow regeneration rate after PBSCT.

[Laser acupuncture therapy for pain and vague complaints in neurosurgery].

The value of diode laser acupuncture for pain and other vague neurological complaints has been studied in chronic patients of head trauma and of cerebrovascular disease or neuralgia in head and neck regions. Gallium-Aluminium-Arsenide (Ga-Al-As) diode laser pain attenuator PANALAS-4000 manufactured by Japan Medical Laser Laboratory was used in the following specifications; wave length: 830 nm, power: 20-40 mW, frequency: 5-50 c.p.s., pulse duty: 0.9. The laser acupuncture was applied to some of the representative Chinese meridian points, local pain points and related nerves for 1 to 7 times in a week. It was given in 310 patients in our neurosurgical department. 175 of 227 patients who complained of pain and 12 of 39 patients who complained of numbness showed improvement within 24 hours, another 24 patients with pain and 16 patients with numbness showed improvement after 24 hours. No side effect was noticed. The duration of the effectiveness was about 2 or 3 days. It is very useful to apply the laser acupuncture for pain and vague complaints in neurosurgery.

Capillary GC/MS determination of organic sulfur compounds detected in oyster and mussel caught in the sea as an oil pollution index.

The organic sulfur compounds usually contained in crude oil were used as a marker of oil pollution in shellfish. The oyster (Crassostrea gigas) and mussel (Mytilus edulis) were caught in the Seto Inland Sea of Japan. Capillary-column gas chromatography/mass spectrometry (GC/MS) of extracts showed the presence of organic sulfur compounds of dibenzothiophene and alkyl dibenzothiophenes (C1-C3). The concentration factor of organic sulfur compounds in the oyster and mussel was determined by gas chromatography using a flame photometric detector.