Analogous mechanisms compensate for neural delays in the sensory and the motor pathways: evidence from motor flash-lag

Motor behaviors require animals to coordinate neural activity across different areas within their motor system. In particular, the significant processing delays within the motor system must somehow be compensated for. Internal models of the motor system, in particular the forward model, have emerged as important potential mechanisms for compensation. For motor responses directed at moving visual objects, there is, additionally, a problem of delays within the sensory pathways carrying crucial position information. The visual phenomenon known as the flash-lag effect has led to a motion-extrapolation model for compensation of sensory delays. In the flash-lag effect, observers see a flashed item colocalized with a moving item as lagging behind the moving item. Here, we explore the possibility that the internal forward model and the motion-extrapolation model are analogous mechanisms compensating for neural delays in the motor and the visual system, respectively. In total darkness, observers moved their right hand gripping a rod while a visual flash was presented at various positions in relation to the rod. When the flash was aligned with the rod, observers perceived it in a position lagging behind the instantaneous felt position of the invisible rod. These results suggest that compensation of neural delays for time-varying motor behavior parallels compensation of delays for time-varying visual stimulation.     

Purification and characterization of the recombinant Thermus sp. strain T2 alpha-galactosidase expressed in Escherichia coli

The nucleotide sequence of the Thermus sp. strain T2 DNA coding for a thermostable alpha-galactosidase was determined. The deduced amino acid sequence of the enzyme predicts a polypeptide of 474 amino acids (M(r), 53,514). The observed homology between the deduced amino acid sequences of the enzyme and alpha-galactosidase from Thermus brockianus was over 70%. Thermus sp. strain T2 alpha-galactosidase was expressed in its active form in Escherichia coli and purified. Native polyacrylamide gel electrophoresis and gel filtration chromatography data suggest that the enzyme is octameric. The enzyme was most active at 75 degrees C for p-nitrophenyl-alpha-D-galactopyranoside hydrolysis, and it retained 50% of its initial activity after 1 h of incubation at 70 degrees C. The enzyme was extremely stable over a broad range of pH (pH 6 to 13) after treatment at 40 degrees C for 1 h. The enzyme acted on the terminal alpha-galactosyl residue, not on the side chain residue, of the galactomanno-oligosaccharides as well as those of yeasts and Mortierella vinacea alpha-galactosidase I. The enzyme has only one Cys residue in the molecule. para-Chloromercuribenzoic acid completely inhibited the enzyme but did not affect the mutant enzyme which contained Ala instead of Cys, indicating that this Cys residue is not responsible for its catalytic function.     

Role of the Rab GTP-binding protein Ypt3 in the fission yeast exocytic pathway and its connection to calcineurin function

A genetic screen for mutations synthetically lethal with fission yeast calcineurin deletion led to the identification of Ypt3, a homolog of mammalian Rab11 GTP-binding protein. A mutant with the temperature-sensitive ypt3-i5 allele showed pleiotropic phenotypes such as defects in cytokinesis, cell wall integrity, and vacuole fusion, and these were exacerbated by FK506-treatment, a specific inhibitor of calcineurin. Green fluorescent protein (GFP)-tagged Ypt3 showed cytoplasmic staining that was concentrated at growth sites, and this polarized localization required the actin cytoskeleton. It was also detected as a punctate staining in an actin-independent manner. Electron microscopy revealed that ypt3-i5 mutants accumulated aberrant Golgi-like structures and putative post-Golgi vesicles, which increased remarkably at the restrictive temperature. Consistently, the secretion of GFP fused with the pho1(+) leader peptide (SPL-GFP) was abolished at the restrictive temperature in ypt3-i5 mutants. FK506-treatment accentuated the accumulation of aberrant Golgi-like structures and caused a significant decrease of SPL-GFP secretion at a permissive temperature. These results suggest that Ypt3 is required at multiple steps of the exocytic pathway and its mutation affects diverse cellular processes and that calcineurin is functionally connected to these cellular processes.     

Interruption of signal transduction between G protein and PKC-ε underlies the impaired myocardial response to ischemic preconditioning in postinfarct remodeled hearts

We have recently shown that the protective mechanism of ischemic preconditioning (PC) is impaired in the myocardium that survived infarction and underwent postinfarct ventricular remodeling. In this study, we examined the hypothesis that failure of PC to activate PKC- underlies the refractoriness of the remodeling heart to PC. Circumflex coronary arteries were ligated in rabbits to induce infarction and subsequent ventricular remodeling, and only sham operations were performed in controls. Hearts were isolated before (i.e. 4 days later) or after (i.e. 2 weeks later) remodeling of the left ventricle and used for isolated buffer-perfused heart experiments. Myocardial infarction was induced in isolated hearts by 30 min global ischemia/2 h reperfusion, and its size was measured by tetrazolium staining. Using separate groups of hearts, tissue biopsies were taken before and after PC, and PKC translocation was assessed by Western blotting. Areas infarcted in vivo by coronary ligation (CL) were excluded from subsequent infarct size/PKC analyses. In the hearts 4 days after CL, PC with 2 cycles of 5 min ischemia/5 min reperfusion induced PKC- translocation from cytosol to particulate fractions and limited infarct size to 40% of control value. In the hearts remodeled 2 weeks after CL, PC failed to induce PKC- translocation and infarct size limitation. In this group, PKC activity and hemodynamic responses to adenosine were similar to those in sham-operated controls. When remodeling after CL was prevented by valsartan infusion (10 mg/kg/day), an angiotensin II type 1 (AT₁) receptor blocker, PC could induce both infarct limitation and PKC- translocation. The present results suggest that persistent activation of AT₁ receptors during remodeling disturbed the PC signaling between G proteins and PKC-, which underlies the refractoriness of the remodeled myocardium to PC.     

Syntheses of 4-methylumbelliferyl-beta-D-xylobioside and 5-bromo-3-indolyl-beta-D-xylobioside for sensitive detection of xylanase activity on agar plates

4-Methylumbelliferyl-beta-D-xylobioside (MU-X2) and 5-bromo-3-indolyl-beta-D-xylobioside (BI-X2) were synthesized as substrates for the detection of xylanase activity on agar plates. A family F/10 xylanase from Streptomyces olivaceoviridis E-86 (FXYN) was able to be more sensitively detected than RBB-xylan by using MU-X2 as a substrate. A mutant xylanase E128H/FXYN having only 1/1000 of the activity of FXYN was also able to be detected on the MU-X2 plate but was not detected on the RBB-xylan plate. A family G/11 xylanase from Streptomyces lividans 66 (Xyn B) was not detected on the MU-X2 plate, but it was able to be detected on the RBB-xylan plate, suggesting that the MU-X2 substrate is specific to family F/10 xylanases. However, none of the xylanases were detected effectively by using BI-X2 as a substrate.     

The presence of a glycosyl phosphatidylinositol-anchored alpha-mannosidase in boar sperm

alpha-Mannosidase and beta-galactosidase were released from boar sperm into the medium by treatment with calcium ionophore A23187 or by 0.2% Brij-35/2% acetic acid. About half as much alpha-mannosidase activity as that in the acid extract was recovered by digestion with phosphatidylinositol-specific phospholipase C (PI-PLC), whereas the liberation rate of beta-galactosidase treated with PI-PLC was low. These results suggest that some alpha-mannosidase is anchored in the plasma membrane of the acrosomal region by attachment to the lipid phosphatidylinositol and that beta-galactosidase is localized mainly in the acrosome or integrated in the plasma membrane by a spanning stretch of hydrophobic peptides. beta-Galactosidase, which is present as an oligomers in the acid extract of sperm, dissociated into monomers under weakly alkaline conditions; under acidic conditions, the monomers associated again. No pH-sensitive association-dissociation of alpha-mannosidase was observed.     

Phytochrome regulation of nuclear gene expression in plants

The photoregulation of gene expression in higher plants was extensively studied during the 1980s, in particular the light-responsive cis -acting elements and trans -acting factors of the Lhcb and rbcS genes. However, little has been discovered about: (1) which plant genes are regulated by light, and (2) which photoreceptors control the expression of these genes. In the 1990s, the functional analysis of the various photoreceptors has progressed rapidly using photoreceptor-deficient mutants, including those of the phytochrome gene family. More recently however, advanced techniques for gene expression analysis, such as fluorescent differential display and DNA microarray technology, have become available enabling the global identification of genes that are regulated by particular photoreceptors. In this paper we describe distinct and overlapping effects of individual phytochromes on gene expression in Arabidopsis thaliana.     

Establishment of an epidermal growth factor-dependent, multipotent neural precursor cell line

Summary We have established a multipotent clonal cell line, named MEB5, from embryonic mouse forebrains after the infection of a retrovirus carrying E7 oncogene of human papillomavirus type 16. MEB5 cells proliferated in serum-free, epidermal growth factor (EGF)-supplemented medium. They expressed markers for neural precursor cells (nestin, A2B5, and RC1) and did not express markers for neurons (class III β-tubulin), astrocytes (glial fibrillary acidic protein), and oligodendrocytes (galactocerebroside). MEB5 cells were stably maintained in an undifferentiated state with a diploid karyotype in the presence of EGF. When they were deprived of EGF, about 50% of the cells died due apoptosis within 24 h. The remaining cells differentiated into neurons, astrocytes, or oligodendrocytes within 2 wk. The newly developed cells with neuronal morphology were immunoreactive for γ-aminobutyric acid and exhibited neuronal electrophysiological properties. When MEB5 cells were treated with leukemia inhibitory for 7 d, they were induced to differentiate exclusively into astrocytes. These results inducate that MEB5 is a cell line with characteristics of EGF-dependent, multipotent neural precursor cells. This cell line should provide a good model system to study the mechanisms of survival, proliferation, and differentiation of the multipotent precursor cells in the central nervous system.