Association of frabin with the actin cytoskeleton is essential for microspike formation through activation of Cdc42 small G protein.

We have recently isolated a novel actin filament-binding protein, named frabin. Frabin has one actin filament-binding domain (ABD), one Dbl homology domain (DHD), first pleckstrin homology domains (PHD) adjacent to DHD, one cysteine rich-domain (CRD), and second PHD from the N terminus to the C terminus in this order. Full-length frabin induces microspike formation and c-Jun N-terminal kinase (JNK) activation. We found here that the fragment of frabin containing DHD and first PHD stimulated guanine nucleotide exchange of Cdc42Hs small G protein, but not that of RhoA or Rac1 small G protein. However, this fragment of frabin did not induce microspike formation, and ABD was additionally necessary for microspike formation. Frabin having ABD was associated with the actin cytoskeleton, whereas frabin lacking ABD was diffusely distributed in the cytoplasm. In contrast, ABD was not necessary for JNK activation but CRD and second PHD were additionally necessary for this activation. These results indicate that the association of frabin with the actin cytoskeleton is essential for microspike formation but not for JNK activation and that different domains of frabin are involved in microspike formation and JNK activation through Cdc42 activation.

Rab11BP/Rabphilin-11, a downstream target of rab11 small G protein implicated in vesicle recycling.

Rab11 small G protein has been implicated in vesicle recycling, but its upstream regulators or downstream targets have not yet been identified. We isolated here a downstream target of Rab11, named rabphilin-11, from bovine brain. Moreover, we isolated from a rat brain cDNA library its cDNA, which encoded a protein with a M(r) of 100,946 and 908 amino acids (aa). Rabphilin-11 bound GTP-Rab11 more preferentially than GDP-Rab11 at the N-terminal region and was specific for Rab11 and inactive for other Rab and Rho small G proteins. Both GTP-Rab11 and rabphilin-11 were colocalized at perinuclear regions, presumably the Golgi complex and recycling endosomes, in Madin-Darby canine kidney cells. In HeLa cells cultured on fibronectin, both the proteins were localized not only at perinuclear regions but also along microtubules, which were oriented toward membrane lamellipodia. Treatment of HeLa cells with nocodazole caused disruption of microtubules and dispersion of GTP-Rab11 and rabphilin-11. Overexpression of the C-terminal fragment of rabphilin-11 (aa 607-730), lacking the GTP-Rab11 binding domain, in HeLa cells reduced accumulation of transferrin at perinuclear regions and cell migration. Rabphilin-11 turned out to be a rat counterpart of recently reported bovine Rab11BP. These results indicate that rabphilin-11 is a downstream target of Rab11 which is involved in vesicle recycling.

Interaction of radixin with Rho small G protein GDP/GTP exchange protein Dbl.

The Rho small G protein family, consisting of the Rho, Rac, and Cdc42 subfamilies, regulates various actin cytoskeleton-dependent cell functions. The Rho subfamily members regulate ERM (ezrin, radixin and moesin)-dependent association of the actin cytoskeleton with the plasma membrane. Moreover, the N-terminal regions of ERM interact with Rho GDI, an inhibitory regulator of all the Rho family members, and reduce its inhibitory action, finally initiating the activation of the Rho family members. We show here that the N-terminal region of radixin furthermore interacts with Dbl, a stimulatory GDP/GTP exchange protein of the Rho family members. This interaction does not affect the Dbl activity to stimulate the GDP/GTP exchange reaction of RhoA, a member of the Rho subfamily. Dbl does not interact with radixin which is precomplexed with Rho GDI, and Rho GDI displaces Dbl from radixin. Thus, radixin plays an important role in activation of the Rho family members by recruiting their positive and negative regulators.

Interactions of drebrin and gephyrin with profilin.

Profilin is an actin monomer-binding protein which stimulates actin polymerization. Recent studies have revealed that profilin interacts with VASP, Mena, Bnilp, Bnrlp, and mDia, all of which have the proline-rich domain. Here, we isolated three profilin-binding proteins from rat brain cytosol by glutathione S-transferase-profilin affinity column chromatography and identified them as Mena, drebrin, and gephyrin. These proteins had a proline-rich domain and directly interacted with profilin.

Family clusters of pulmonary tuberculosis in a suburban area of Japan.

A survey was conducted in a metropolitan suburb to examine family clusters of pulmonary tuberculosis patients and related factors. Firstly, a descriptive study was done based on the review of tuberculosis registration cards over a 11-yr period. Of 1106 cases, 109 (9.9%) were found to occur in households which had already had one or more pulmonary tuberculosis patient during the period. The most affected were four- and eight-member families with infants and adolescents accounting for most of the second or later patients. Secondly, a home visit survey was performed to investigate factors associated with family clusters of phthisis. Results showed that significant variables were the length of patient's delay (interval between perception of physical symptoms and visit to the physician), the number of rooms, and lack of self-motivation to seek treatment. Log-linear analyses revealed interaction among these variables.

Ischemic injury in cirrhotic livers: an experimental study of the temporary arrest of hepatic circulation.

In order to prevent massive bleeding at hepatectomy, the temporary arrest of hepatic circulation is often performed but it is not clear whether this arrest of circulation is tolerated equally by the cirrhotic liver and the normal liver. We investigated biochemically the detailed effects of ischemia on cirrhotic livers in rats with experimentally induced liver cirrhosis. Thioacetoamide was used to prepare the rat cirrhotic liver model (LC group, n = 6). After laparotomy, the vessels to the left lateral lobe were clamped for 30 min and then declamped. Changes in AST isozymes and the aminogram in the blood were examined after ischemia. Postischemic changes in hepatic adenine nucleotides (AdN) and the brain aminogram were also examined. These were compared with those of normal liver ischemia (N group, n = 6) at 24 hr after recirculation. In the LC group, serum levels of mitochondrial AST, a parameter of necrotic cells, were significantly higher than those of the N group. Hepatic AdN levels decreased to 60.6% of the original levels after ischemic injury but those of the N group remained at 95.4% of the original level. Since AdN in tissue is accepted as a reliable parameter of viable cells, cirrhotic livers subjected to ischemia might have more necrotic cells than normal livers. Sequential analysis of serum aminograms of the LC group after ischemia revealed that the ratio of Val+Leu+Ileu/Tyr+Phe decreased to near 1.0 but that of the N group always remained higher than 3.0. Based on these results, it was concluded that ischemic injury in cirrhotic livers is more hazardous than that in normal livers.

Possible involvement of presynaptic 5-HT autoreceptors in effect of lithium on 5-HT release in hippocampus of rat.

The effects of subacute treatment with lithium on the stimulation-induced release of serotonin (5-HT) and the function of 5-HT autoreceptors in the hippocampus and frontal cortex of the rat were studied. In the rats treated with lithium for 3 days, the high K+-evoked release of endogenous 5-HT from the slices of hippocampus, but not the slices of frontal cortex, was significantly increased. High K+-induced release of [3H]5-HT from the slices of hippocampus of control rats preloaded with [3H]5-HT was decreased when the slices were exposed to 5-HT, while it was increased when they were exposed to methiothepin. In the slices of hippocampus from the lithium-treated rats, this inhibitory effect of 5-HT on the release of [3H]5-HT, evoked by either high K+ or electrical stimulation was significantly attenuated. On the other hand, it was not modified in the slices of frontal cortex. The results suggest that lithium may inhibit the function of 5-HT autoreceptors to regulate the release of 5-HT. This action may, in part, trigger the development of the down-regulation of 5-HT1 receptors occurring in the hippocampus but not in the frontal cortex after chronic treatment with lithium.

Lithium decreases 5-HT1 receptors but increases 5-HT-sensitive adenylate cyclase activity in rat hippocampus.

The effect of lithium (Li) treatment on serotonin1 (5-HT1) receptors and 5-HT-sensitive adenylate cyclase (ACase) activity in rat hippocampus was studied. [3H]5-HT binding to 5-HT1 receptors was decreased after either subacute (5 days) or chronic (3 weeks) treatment. In contrast, 5-HT-stimulated [3H]cyclic adenosine monophosphate ([3H]cAMP) formation was unchanged after 5 days of Li treatment, but was increased after 3 weeks of treatment. There was no difference in the inhibitory effects of guanosine triphosphate (GTP) on [3H]5-HT binding between the subacute Li and control groups. In addition, [3H]cAMP formation induced by GTP was not changed in the subacute group, whereas chronic treatment decreased it. These results suggested that chronic Li treatment caused the facilitation of 5-HT-sensitive ACase activity in spite of a decrease in the density of 5-HT1 receptors in the hippocampus. The enhancement of the coupling between receptor and ACase seemed not to be involved in these Li effects.

[Effect of long-term lithium treatment on serotonin syndrome in rats].

Brain 5-HT receptors are reported to play an important role in the therapeutic effect of Li in manic-depressive illness. In the present study we examined the effect of Li on 5-HT syndrome which is postulated to be the physiological response of 5-HT1 receptors. Intraperitoneal injection of the 5-HT receptor agonist 5-MeODMT elicited 5-HT syndrome in a dose-dependent manner. Then the incidence of 5-HT syndrome elicited by 2 mg/kg of 5-MeODMT was measured in animals under long-term Li treatment. Four out of six abnormal behaviors of the 5-HT syndrome, i.e. forepaw treading, resting tremor, rigidity, and head weaving, were significantly increased by the 9-day treatment with Li. On the 17th day of Li treatment, the incidence of all six behaviors, including hindlimb abduction and straub tail, were significantly enhanced. This Li-induced supersensitivity of 5-HT1 receptor-mediated behaviors inconsistent with the previous reports of the down-regulation of 5-HT1 receptors measured by receptor binding assay. The reason for this discrepancy is obscure. Further examination of the effect of Li on the signal-transduction system from the receptor to the behavior will be necessary.

Long-term lithium treatment causes serotonin receptor down-regulation via serotonergic presynapses in rat brain.

The effects of lithium treatment on serotonin (5-HT) receptors in rat frontal cortex and hippocampus were investigated. Long-term lithium treatment strongly blocked 5-hydroxytryptophan-induced head twitches, while acute lithium administration by itself induced head twitches in rats, and ketanserin blocked this acute lithium action. Long-term administration of lithium decreased the number of not only 5-HT2 receptors in the frontal cortex but also 5-HT1 and 5-HT2 receptors in the hippocampus in rats. Decreases in 3H-5-HT binding to hippocampal 5-HT1 receptors and 3H-spiperone binding to frontal cortical 5-HT2 receptors, caused by chronic lithium treatment, were abolished by co-administration of p-chlorophenylalanine, and were enhanced by co-administration with methiothepin. The turnover of 5-HT in either frontal cortex or hippocampus was facilitated by lithium, and co-administered methiothepin enhanced this facilitation. These results suggest that long-term lithium treatment causes the down-regulation of postsynaptic 5-HT1 and 5-HT2 receptors, in part probably through its action on presynaptic nerve terminals.

The active principles of the branchlet and hook of Uncaria sinensis Oliv. examined with a 5-hydroxytryptamine receptor binding assay.

Of the alkaloids obtained from Uncaria sinensis Oliv., geissoschizine methyl ether, corynantheine and dihydrocorynantheine decreased specific [3H]5-HT binding to membrane preparations from rat brain and from in-vitro experiments on guinea-pig ileum, these alkaloids were found to be partial agonists for 5-HT receptors. Therefore, they might be useful in the treatment of diseases resulting from disorders of 5-HT metabolism.