The antiataxic mechanism of TRH in rolling mouse Nagoya: the effects of pretreatment with dopaminergic and cholinergic drugs

Antiataxic mechanisms were investigated in Rolling mouse Nagoya (RMN). The present study was to elucidate the influence of dopaminergic (pimozide, apomorphine) and cholinergic (atropine, physostigmine) drugs on the antiataxic effect of TRH. The degree of ataxic gait and spontaneous motor activities in RMN were measured by the open field method and ANIMEX-II Pretreatment with pimozide and apomorphine had no influence on the antiataxic effects of TRH, while pretreatment with physostigmine suppressed these effects and in contrast, with atropine, increased then. The increase of spontaneous motor activities after TRH injection was antagonized by pretreatment with pimozide and physostigmine, but accentuated by pretreatment with atropine. These results may indicate that the antiataxic effects of TRH are, at least partially, mediated by the cholinergic mechanism.     

A novel monoclonal antibody, RM-4, specifically recognizes rat macrophages and dendritic cells in formalin-fixed, paraffin- embedded tissues

Summary An anti-rat macrophage/dendritic cell monoclonal antibody, RM-4, was produced using a homogenate of silica-induced lung granulomas of rat as immunogen. Immunohistochemistry demonstrated that RM-4 was specific for macrophage and dendritic cell populations residing in various organs and tissues. It did not react with any cells other than macrophage/dendritic cells. In the double staining of the spleen, RM-4-positive macrophages showed wider distribution than those of the four other anti-rat macrophage monoclonal antibodies compared. The immunoreactivity of RM-4 was well preserved not only in frozen sections but also in formalin-fixed, paraffin-embedded tissues. The isotype of the monoclonal antibody was IgG₁ kappa and its antigen molecular weight was 46 kDa. Immunoelectron microscopy revealed positive reaction products for RM-4 on the membrane of endosomes and lysosomes in macrophages and epidermal Langerhans cells. Reaction intensity increased after thioglycolate elicitation or endocytosis regardless of ingested materials. From these data, it is concluded that RM-4 recognizes a membrane protein of endolysomes in macrophages and dendritic cells. The antigen may play a role in endolysosomal processing. RM-4 is considered to be a useful tool not only for identifying macrophage/dendritic cells both in frozen and paraffin-embedded tissues, but also for evaluating their endolysosomal processing. This revised version was published online in November 2006 with corrections to the Cover Date.     

Synthesis of acylated SOD derivatives which bind to the biomembrane lipid surface and dismutate extracellular superoxide radicals

Involvement of oxygen radicals in the pathogenesis of various inflammatory diseases has been the focus of recent attention. Since lipid peroxidation of cell membranes is postulated to be one of the major reasons for radical-induced tissue injury, inhibition of oxygen toxicity at or near plasma membranes is important. To metabolize extracellular superoxide radicals effectively at or near cell membranes, we synthesized amphipathic superoxide dismutase (SOD) derivatives (AC-SOD) by covalently linking hydrophobic fatty acids with different chain lengths, such as caprylic acid, capric acid, lauric acid and myristic acid, to the lysyl amino groups of the enzyme. When incubated with erythrocytes or polymorphonuclear leukocytes (PMNs), AC-SOD, but not SOD, bound to plasma membranes of these cells. When topically instilled to the eye, AC-SOD also bound to corneal epithelial cell surface. Upon activation by phorbolmyristyl acetate, extracellular cytochrome c was rapidly reduced by PMNs which were pretreated with SOD. In contrast, PMNs preincubated with AC-SOD failed to catalyze the reduction of cytochrome c under the same experimental conditions. These results suggested that AC-SOD bound to cell membranes and effectively dismutated superoxide radicals at or on the outer surface of plasma membranes.     

Identification and Determination of Selenoneine, 2-Selenyl-N α , N α , N α -Trimethyl-l-Histidine, as the Major Organic Selenium in Blood Cells in a Fish-Eating Population on Remote Japanese Islands

Selenoneine is the major selenium compound in fish muscles, and fish appears to be an important source of selenium in the fish-eating population. Selenoneine has strong antioxidant activity and a detoxifying function against methylmercury (MeHg) toxicity. Dietary intake, bioaccumulation, and metabolism of selenoneine have not been characterized in humans. A nutritional survey was conducted in remote islands of the Kagoshima Prefecture in Japan. To evaluate the potential risks and benefits of fish consumption for health, we measured concentrations of selenoneine, total selenium, MeHg, inorganic mercury, and polyunsaturated fatty acid (LC-PUFA) in the blood of a fish-eating human population. The erythrocyte, leukocyte, and platelet residues following removal of serum (cellular fraction) contained 0.510 μg Se/g, 0.212 μg selenoneine Se/g, and 0.262 μg Se-containing proteins Se/g, whereas the serum contained 0.174 μg total Se/g. Selenoneine was highly concentrated in the cellular fraction in a manner that was dependent on subjects' frequency of fish consumption. Concentrations of selenoneine were closely correlated with concentrations of MeHg in the cellular fraction. Selenoneine is the major chemical form of selenium in the blood cells of this fish-eating human population and may be an important biomarker for selenium redox status.     

Classification of Self-Driven Mental Tasks from Whole-Brain Activity Patterns

During wakefulness, a constant and continuous stream of complex stimuli and self-driven thoughts permeate the human mind. Here, eleven participants were asked to count down numbers and remember negative or positive autobiographical episodes of their personal lives, for 32 seconds at a time, during which they could freely engage in the execution of those tasks. We then examined the possibility of determining from a single whole-brain functional magnetic resonance imaging scan which one of the two mental tasks each participant was performing at a given point in time. Linear support-vector machines were used to build within-participant classifiers and across-participants classifiers. The within-participant classifiers could correctly discriminate scans with an average accuracy as high as 82%, when using data from all individual voxels in the brain. These results demonstrate that it is possible to accurately classify self-driven mental tasks from whole-brain activity patterns recorded in a time interval as short as 2 seconds.     

Involvement of protein kinase C in the regulation of assembly-disassembly of neurofilaments in vitro

Protein kinase C phosphorylated the major mammalian neurofilament protein (NF-L) with approximately 3 mol phosphate per mol protein. The phosphorylated NF-L no longer formed the filaments. Sequential analysis of the tryptic phosphopeptides, together with the known primary sequence, revealed that Ser-12, Ser-27, Ser-33 and Ser-51 were phosphorylated by protein kinase C. These findings contribute toward elucidation of mechanisms regulating the functions of neurofilaments.     

Phosphorylation by actin kinase of the pointed end domain on the actin molecule.

Fragmin from plasmodium of Physarum polycephalum binds G-actin and severs F-actin in the presence of Ca2+ over 10(-6) M. The fragmin-actin complex consisting of fragmin and G-actin nucleates actin polymerization and caps the barbed (fast growing) end of F-actin, regardless of the concentrations of Ca2+, and the actin filaments are shortened. Actin kinase purified from plasmodium abolishes the nucleation and capping activities of the complex by phosphorylating actin of the fragmin-actin complex (Furuhashi, K., and Hatano, S. (1990) J. Cell. Biol. 111, 1081-1087). This inactivation of the complex leads to production of long actin filaments. We obtained evidence that Physarum actin is phosphorylated by actin kinase at Thr-201, and probably at Thr-202 and/or Thr-203, with 1 mol of phosphate distributed among them. This finding raises the possibility that the site of phosphorylation, Thr-201 to Thr-203, is positioned on the pointed (slow growing) end domain of the actin molecule, because growth of actin filaments from the fragmin-actin complex occurs only from the pointed end. These observations are consistent with a model of the three-dimensional structure of G-actin. Inactivation of the fragmen-actin complex may follow phosphorylation of the pointed end domain of actin.     

A modified theta-toxin produced by limited proteolysis and methylation: a probe for the functional study of membrane cholesterol.

A derivative of cytolytic theta-toxin from Clostridium perfringens was prepared by limited proteolytic digestion of the native toxin followed by methylation. Among the chloroform/methanol-extractable, lipid components of sheep and human erythrocytes, the proteinase-nicked and methylated derivative (MC theta) specifically binds to cholesterol. While MC theta retains binding affinity comparable to that of intact toxin, it causes no obvious membrane damage, resulting in no hemolysis at temperatures of 37 degrees C or lower. Using MC theta, we demonstrated the possible existence of high- and low-affinity sites for theta-toxin on sheep erythrocytes at both 37 degrees C and 10 degrees C. The number of high-affinity sites on sheep erythrocytes was estimated to be approximately 3-times larger at 37 degrees C than that at 10 degrees C. In addition, high- and low-affinity sites were demonstrated in human erythrocytes and a lymphoma B cell line, BALL-1 cells. Both binding sites disappear upon simultaneous treatment of cells with sublytic doses of digitonin, suggesting that cholesterol is an essential component of both the high- and low-affinity sites and that the mode of cholesterol existence in plasma membranes is heterogeneous in these cells. Because of its high affinity for membrane cholesterol without causing any obvious membrane changes at physiological temperatures, MC theta may provide a probe for use in the functional study of membrane cholesterol.