NF-M is an essential target for the myelin-directed "outside-in" signaling cascade that mediates radial axonal growth

Neurofilaments are essential for acquisition of normal axonal calibers. Several lines of evidence have suggested that neurofilament-dependent structuring of axoplasm arises through an "outside-in" signaling cascade originating from myelinating cells. Implicated as targets in this cascade are the highly phosphorylated KSP domains of neurofilament subunits NF-H and NF-M. These are nearly stoichiometrically phosphorylated in myelinated internodes where radial axonal growth takes place, but not in the smaller, unmyelinated nodes. Gene replacement has now been used to produce mice expressing normal levels of the three neurofilament subunits, but which are deleted in the known phosphorylation sites within either NF-M or within both NF-M and NF-H. This has revealed that the tail domain of NF-M, with seven KSP motifs, is an essential target for the myelination-dependent outside-in signaling cascade that determines axonal caliber and conduction velocity of motor axons.     

Light Activation of NADP-Malate Dehydrogenase in Guard Cell Protoplasts from Vicia faba L

Light-induced swelling of guard cell protoplasts (GCP) from Vicia faba was accompanied by increases in content of K⁺ and malate. DCMU inhibited the increase of K⁺ and malate, and consequently swelling.

Effect of light on the activity of selected enzymes that take part in malate formation was studied. When isolated GCP were illuminated, NADP-malate dehydrogenase (NADP-MDH) was activated, and the activity reached a maximum within 5 minutes. The enzyme activity underwent 5- to 6-fold increase in the light. Upon turning off the light, the enzyme was inactivated in 5 minutes NAD-MDH and phosphoenolpyruvate carboxylase (PEPC) were not influenced by light. The rapid light activation of NADP-MDH was inhibited by DCMU, suggesting that the enzyme was activated by reductants from the linear electron transport in chloroplasts. An enzyme localization study by differential centrifugation indicates that NADP-MDH is located in the chloroplasts, NAD-MDH in the cytosol and mitochondria, and PEPC in the cytosol. After light activation, the activity of NADP-MDH in guard cells was 10 times that in mesophyll cells on a chlorophyll basis. The physiological significance of light-dependent activation of NADP-MDH in guard cells is discussed in relation to stomatal movement.

     

Gene replacement in mice reveals that the heavily phosphorylated tail of neurofilament heavy subunit does not affect axonal caliber or the transit of cargoes in slow axonal transport

The COOH-terminal tail of mammalian neurofilament heavy subunit (NF-H), the largest neurofilament subunit, contains 44-51 lysine-serine-proline repeats that are nearly stoichiometrically phosphorylated after assembly into neurofilaments in axons. Phosphorylation of these repeats has been implicated in promotion of radial growth of axons, control of nearest neighbor distances between neurofilaments or from neurofilaments to other structural components in axons, and as a determinant of slow axonal transport. These roles have now been tested through analysis of mice in which the NF-H gene was replaced by one deleted in the NF-H tail. Loss of the NF-H tail and all of its phosphorylation sites does not affect the number of neurofilaments, alter the ratios of the three neurofilament subunits, or affect the number of microtubules in axons. Additionally, it does not reduce interfilament spacing of most neurofilaments, the speed of action potential propagation, or mature cross-sectional areas of large motor or sensory axons, although its absence slows the speed of acquisition of normal diameters. Most surprisingly, at least in optic nerve axons, loss of the NF-H tail does not affect the rate of transport of neurofilament subunits.     

Filipin resistance in intermediate junction membranes of guinea pig ependyma: possible relationship to filamentous underlying

Plasma membranes in intermediate junctions of ependymal cells are found to show considerable resistance to the antibiotic filipin, suggesting low cholesterol in these membranes. Further, ependymal cells were treated with cytochalasin B (CB) infused into the cerebral ventricle in vivo, and then incubated with filipin. When treated with CB, intermediate junctions show a decrease in their underlying density, mainly composed of microfilaments, and their membranes are found to be more affected by filipin. This reduction of resistance to the antibiotic is clearly demonstrated by thin-section and freeze-fracture as well as quantitative analysis. Nonjunctional lateral membranes, however, show no significant difference in the degree of filipin effect whether treated with CB or not. Although biochemical data on lipid composition have not been available for the intermediate junction membranes, we bring forward a possibility that resistance to filipin in these membranes may come not from less cholesterol but from morphological membrane stability brought about by the filamentous underlying.     

Effect of a warm‐up sample on stabilizing the performance of untrained panelists in time–intensity evaluation

To improve the reliability of sensory evaluation, it is necessary to minimize response bias. In this study, we investigated the effect of a warm‐up sample on time–intensity evaluation of after‐flavor. Untrained panelists evaluated continuously perceived intensity of bitterness and retronasal aroma, using four types of samples (two brands of coffee beverages × two types of lids). Half of participants evaluated bitterness intensity in the first session and retronasal aroma intensity in the second session, whereas the other half evaluated them in the opposite order. Each session consisted of four trials, and we regarded the first trial sample of each session as a warm‐up. Results revealed that perceived intensity was significantly lower in the first trial than in the following trials, and that the time courses of the second, third, and fourth trials were similar except when participants evaluated bitterness intensity in the second session. In other words, the use of a warm‐up sample stabilized the performance of time–intensity evaluation of bitterness and retronasal aroma in the first session. Furthermore, no warm‐up sample was needed in the second session when participants evaluated bitterness intensity, but a warm‐up was needed when they evaluated retronasal aroma intensity.

Practical application

Based on the results of this study, we concluded that the use of a warm‐up sample stabilized performance in time–intensity evaluation of after‐flavor by untrained panelists. We proposed that in order to obtain reliable performance in time–intensity evaluation of after‐flavor, untrained panelists should be provided a training trial using warm‐up sample before staring the test trials.     

Phenotype-dependent expression of alpha-smooth muscle actin in visceral smooth muscle cells

Alpha-Smooth muscle actin is one of the molecular markers for a phenotype of vascular smooth muscle cells, because the actin is a major isoform expressed in vascular smooth muscle cells and its expression is upregulated during differentiation. Here, we first demonstrate that the phenotype-dependent expression of this actin in visceral smooth muscles is quite opposite to that in vascular smooth muscles. This actin isoform is not expressed in adult chicken visceral smooth muscles including gizzard, trachea, and intestine except for the inner layer of intestinal muscle layers, whereas its expression is clearly detected in these visceral smooth muscles at early stages of the embryo (10-day-old embryo) and is developmentally downregulated. In cultured gizzard smooth muscle cells maintaining a differentiated phenotype, alpha-smooth muscle actin is not detected while its expression dramatically increases during serum-induced dedifferentiation. Promoter analysis reveals that a sequence (-238 to -219) in the promoter region of this actin gene acts as a novel negative cis-element. In conclusion, the phenotype-dependent expression of alpha-smooth muscle actin would be regulated by the sum of the cooperative contributions of the negative element and well-characterized positive elements, purine-rich motif, and CArG boxes and their respective transacting factors.     

Light-dependent K(+) channels in the mollusc Onchidium simple photoreceptors are opened by cGMP

Light-dependent K(+) channels underlying a hyperpolarizing response of one extraocular (simple) photoreceptor, Ip-2 cell, in the marine mollusc Onchidium ganglion were examined using cell-attached and inside-out patch-clamp techniques. A previous report (Gotow, T., T. Nishi, and H. Kijima. 1994. Brain Res. 662:268-272) showed that a depolarizing response of the other simple photoreceptor, A-P-1 cell, results from closing of the light-dependent K(+) channels that are activated by cGMP. In the cell-attached patch recordings of Ip-2 cells, external artificial seawater (ASW) was replaced with a modified ASW containing 150 mM K(+) and 200 mM Mg(2+) to suppress any synaptic input and to maintain the membrane potential constant. When Ip-2 cells were equilibrated with this modified ASW, the internal K(+) concentration was estimated to be 260 mM. Light-dependent single-channels in the cell-attached patch on these cells were opened by light but scarcely by voltage. After confirming the light-dependent channel activity in the cell-attached patches, an application of cGMP to the excised inside-out patches newly activated a channel that disappeared on removal of cGMP. Open and closed time distributions of this cGMP-activated channel could be described by the sum of two exponents with time constants tau(o1), tau(o2) and tau(c1), tau(c2), respectively, similar to those of the light-dependent channel. In both the channels, tau(o1) and tau(o2) in ms ranges were similar to each other, although tau(c2) over tens of millisecond ranges was different. tau(o1), tau(o2), and the mean open time tau(o) were both independent of light intensity, cGMP concentration, and voltage. In both channels, the open probability increased as the membrane was depolarized, without changing any of tau(o2) or tau(o). In both, the reversal potentials using 200- and 450-mM K(+)-filled pipettes were close to the K(+) equilibrium potentials, suggesting that both the channels are primarily K(+) selective. Both the mean values of the channel conductance were estimated to be the same at 62 and 91 pS in 200- and 450-mM K(+) pipettes at nearly 0 mV, respectively. Combining these findings with those in the above former report, it is concluded that cGMP is a second messenger which opens the light-dependent K(+) channel of Ip-2 to cause hyperpolarization, and that the channel is the same as that of A-P-1 closed by light.     

Selective localization of Bcl-2 to the inner mitochondrial and smooth endoplasmic reticulum membranes in mammalian cells

Bcl-2, an anti-apoptotic protein, is believed to be localized in the outer mitochondrial membrane, endoplasmic reticulum, and nuclear envelope. However, Bcl-2 has also been suggested as playing a role in the maintenance of mitochondrial membrane potential, indicating its possible association with the inner mitochondrial membrane. We therefore further examined the exact localization of Bcl-2 in mitochondria purified from wild-type and bcl-2-transfected PC12 cells and pre- and postnatal rat brains. Double immunostaining demonstrated that Bcl-2 was co-localized with subunit beta of F1F0ATPase in the inner mitochondrial membrane. Biochemical analysis of isolated mitochondria using digitonin and trypsin suggests an association of Bcl-2 with the inner mitochondrial membrane. More interestingly, the majority of Bcl-2 disappeared from the inner membrane of mitochondria when cultured under serum deprivation. These results suggest that Bcl-2 acts as an anti-apoptotic regulator by localizing mainly to the inner mitochondrial and smooth ER membranes.     

Effects of Desmin Gene Knockout on Mice Heart Mitochondria

In heart tissue from mice lacking the intermediate filament (IF) desmin, mitochondria show an abnormal shape and distribution (Thornell et al., 1997). In the present study we have isolated heart mitochondria from desmin null (D–/–) and control (D+/+) mice, and analyzed their composition by SDS–PAGE, immunoblotting, and enzyme measurements. We found both in vitro and in situ that the conventional kinesin, the microtubule-associated plus-end directed motor, was frequently associated with D+/+ heart mitochondria, but not with D–/– heart mitochondria, suggesting that the positioning of mitochondria in heart is a dynamic event involving the IF desmin, the molecular motor kinesin, and, most likely, the microtubules (MT) network. Furthermore, an increased capacity in energy production was found, as indicated by a threefold higher creatine kinase activity in heart mitochondria from D–/– compared to D+/+ mice. We also observed a significantly lower amount of cytochrome c in heart mitochondria from D–/– mice, and a relocalization of Bcl-2, which may indicate an apoptotic condition in the cell leading to the earlier reported pathological events, such as cardiomyocytes degeneration and calcinosis of the heart (Thornell et al., 1997).