Ischemic flow threshold for striatal dopamine release in rats

To determine the level of cerebral blood flow reduction which causes striatal dopamine release, extracellular dopamine and cerebral blood flow was simultaneously determined using in vivo brain dialysis and a hydrogen clearance method, respectively, in the striatum of spontaneously hypertensive rats, before and during experimental cerebral ischemia. The ischemic flow threshold for neurotransmitter dopamine release was found to be 20% of the resting value or 8–10 ml/100g/min of cerebral blood flow, being similar to those for energy and membrane failures.     

Lipoprotein biosynthesis in the larvae of the tobacco hornworm, Manduca sexta

Lipoprotein biosynthesis in larvae of the tobacco hornworm (Manduca sexta) was investigated. By immunoblotting, it was shown that the apoproteins are present in the fat body, but not in the midgut. Fat body incubated in vitro with [35S]methionine secreted labeled apoproteins. However, when the density of the secreted particle was determined, it was found at 1.24-1.28 g/ml instead of 1.15 g/ml, which is the density of the circulating lipoprotein. Lipid analysis of immunoprecipitated lipoprotein secreted by the fat body showed a phospholipid/diacylglycerol ratio of 8.3 rather than 0.9, the ratio found in the circulating lipoprotein. When labeled oleic acid or triolein was fed to larvae, it was found that greater than 98% of the label in the circulating lipoprotein was in diacylglycerol. In studies using animals raised on a fat-free diet, it was shown that the circulating lipoprotein has properties comparable to those of the material secreted in vitro by the fat body and that this diacylglycerol-poor particle can be converted to the normal lipoprotein by feeding a bolus of triolein. These data support the hypothesis that the fat body makes and secretes a "nascent" lipoprotein which contains apoproteins and phospholipid, but is devoid of diacylglycerol. The diacylglycerol is then picked up from the midgut to complete assembly of the mature circulating lipoprotein.     

Ca2+,Mg2+-ATPase of microsomal membranes from bovine aortic smooth muscle. Identification and characterization of an acid-stable phosphorylated intermediate of the Ca2+,Mg2+-ATPase

An acid-stable phosphoprotein was formed in a microsomal membrane fraction isolated from bovine aortic smooth muscle in the presence of Mg2+ + ATP and Ca2+. The microsomes also showed Ca2+ uptake activity. The Ca2+ dependence of phosphoprotein formation and of Ca2+ uptake occurred over the same range of Ca2+ concentration (1-10 microM), and resembled similar findings from rabbit skeletal microsomes. The molecular weight of the phosphorylated protein, estimated by SDS-gel electrophoresis, was approximately 105,000. The phosphoprotein was labile at alkaline pH, and its decomposition was accelerated by hydroxylamine. Half-maximum incorporation of 32P in the presence of 10 microM Ca2+ occurred at 60 nM ATP. The calcium-dependent phosphoprotein formation was not affected by 5 mM NaN3, but was inhibited in a dose-dependent fashion by ADP with a 50% inhibition occurring at 180 microM. Fifty mM MgCl2 was required for the maximal phosphorylation. The rate of phosphoprotein decomposition after adding 2 mM EGTA was accelerated by varying the Mg2+ concentration from 10 microM to 3 mM. Alkaline pH (9.0) slowed the rate of phosphoprotein decay. Optimal Ca2+-dependent phosphoprotein occurred at 15 degrees C over a broad pH range (6.4 to 9.0). The activation energy of EGTA-induced phosphoprotein decomposition was 25.6 kcal/mol between 0 and 16 degrees C and 14.6 kcal/mol between 16 and 30 degrees C. The phosphoprotein formed by aortic microsomes was thus quite similar to the acid-stable phosphorylated intermediate of the Ca2+-transport ATPase of sarcoplasmic reticulum from skeletal and cardiac muscle. These data suggest that the Ca2+-dependent phosphoprotein is a reaction intermediate of the Ca2+,Mg2+-ATPase of the aortic microsomes.     

Immunogold Localization of Ribulose-1,5-Bisphosphate Carboxylase with Reference to Pyrenoid Morphology in Chloroplasts of Synchronized Euglena gracilis Cells

Euglena gracilis strain (Z) cells were synchronized under photoautotrophic conditions using a 14 hour light:10 hour dark regimen. The cells grew during the light period (growth phase) and divided during the following 10 hour period either in the dark or in the light (division phase). Changes in morphology of the pyrenoid and in the distribution of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) within the chloroplasts were followed by immunoelectron microscopy during the growth and division phases of Euglena cells. Epon-embedded sections were labeled with an antibody to the holoenzyme followed by protein A-gold. The immunoreactive proteins were concentrated in the pyrenoid, and less densely distributed in the stroma during the growth phase. During the division phase, the pyrenoid could not be detected and the gold particles were dispersed throughout the stroma. Toward the end of the division phase, the pyrenoid began to form in the center of a chloroplast, and the immunoreactive proteins started to concentrate over that rudimentary pyrenoid. During the growth phase, small areas rich in gold particles, called `satellite pyrenoid,' were observed, in addition to the main pyrenoid. From a comparison of photosynthetic CO₂-fixation with the total carboxylase activity of Rubisco extracted from Euglena cells in the growth phase, it is suggested that the carboxylase in the pyrenoid functions in CO₂-fixation in photosynthesis.     

Demonstration of Antigenic and Genotypic Variation in Orientia tsutsugamushi Which Were Isolated in Japan,and Their Classification into Type and Subtype

A total of 40 strains of Orientia tsutsugamushi (34 isolates from patients and trombiculid mites in Japan, and 6 prototype strains of antigenic variants) were examined for classification based on the reactivities with type-specific monoclonal antibodies in indirect immunofluorescence tests, and on the restriction fragment length polymorphism of a polymerase chain reaction (PCR)-amplified 56-kilodalton type-specific antigenic protein gene. By these methods, several antigenic and genotypic variants were found among the strains, and these variants were classified into types and further into subtypes. These results suggest that there are many variants in O. tsutsugamushi, and the methods used here seem to be useful for the systematic classification of the numerous variants. A strain which may be a new type distinguishable from those identified previously was also found in this study. Furthermore, variety in the degree of pathogenicity in mice related to type and/or subtype classification were observed.     

Correction to: Comparison of the usability of an automatic sleep staging program via portable 1-channel electroencephalograph and manual sleep staging with traditional polysomnography

Sleep and Biological Rhythms -     

The role of p63 in embryonic external genitalia outgrowth in mice

Embryonic external genitalia (genital tubercle [GT]) protrude from the cloaca and outgrow as cloacal development progresses. Individual gene functions and knockout phenotypes in GT development have been extensively analyzed; however, the interactions between these genes are not fully understood. In this study, we investigated the role of p63, focusing on its interaction with the Shh–Wnt/Ctnnb1–Fgf8 pathway, a signaling network that is known to play a role in GT outgrowth. p63 was expressed in the epithelial tissues of the GT at E11.5, and the distal tip of the GT predominantly expressed the ΔNp63α isoform. The GTs in p63 knockout embryos had normal Shh expression, but CTNNB1 protein and Fgf8 gene expression in the distal urethral epithelium was decreased or lost. Constitutive expression of CTNNB1 in p63-null embryos restored Fgf8 expression, accompanied by small bud structure development; however, such bud structures could not be maintained by E13.5, at which point mutant GTs exhibited severe abnormalities showing a split shape with a hemorrhagic cloaca. Therefore, p63 is a key component of the signaling pathway that triggers Fgf8 expression in the distal urethral epithelium and contributes to GT outgrowth by ensuring the structural integrity of the cloacal epithelia. Altogether, we propose that p63 plays an essential role in the signaling network for the development of external genitalia.