Biochemical characterization of the Rho GTPase-regulated actin assembly by diaphanous-related formins, mDia1 and Daam1, in platelets

The diaphanous-related formins are actin nucleating and elongating factors. They are kept in an inactive state by an intramolecular interaction between the diaphanous inhibitory domain (DID) and the diaphanous-autoregulatory domain (DAD). It is considered that the dissociation of this autoinhibitory interaction upon binding of GTP-bound Rho to the GTPase binding domain next to DID induces exposure of the FH1-FH2 domains, which assemble actin filaments. Here, we isolated two diaphanous-related formins, mDia1 and Daam1, in platelet extracts by GTP-RhoA affinity column chromatography. We characterized them by a novel assay, where beads coated with the FH1-FH2-DAD domains of either mDia1 or Daam1 were incubated with platelet cytosol, and the assembled actin filaments were observed after staining with rhodamine-phalloidin. Both formins generated fluorescent filamentous structures on the beads. Quantification of the fluorescence intensity of the beads revealed that the initial velocity in the presence of mDia1 was more than 10 times faster than in the presence of Daam1. The actin assembly activities of both FH1-FH2-DADs were inhibited by adding cognate DID domains. GTP-RhoA, -RhoB, and -RhoC, but not GTP-Rac1 or -Cdc42, bound to both mDia1 and Daam1 and efficiently neutralized the inhibition by the DID domains. The association between RhoA and Daam1 was induced by thrombin stimulation in platelets, and RhoA-bound endogenous formins induced actin assembly, which was inhibited by the DID domains of Daam1 and mDia1. Thus, mDia1 and Daam1 are platelet actin assembly factors having distinct efficiencies, and they are directly regulated by Rho GTPases.     

Collagen XVIII and corneal reinnervation following keratectomy

The significance of collagen XVIII in the regulation of corneal reinnervation remains largely unknown. We used whole-mount immunoconfocal microscopy to localize collagen XVIII to the nerve basement membrane of wild-type (WT) mouse corneas. Transmission electron microscopy showed corneal nerve disorganization in collagen XVIII knockout mice (col18a1(-/-)). Antibody 2H3-specific neurofilament colocalized with collagens XVIII and IV and laminin-2 in WT mouse corneas, but did not colocalize with collagen IV and laminin-2 in col18a1(-/-) mouse corneas. Following keratectomy, col18a1(-/-) mice displayed decreased corneal neurite extension compared to WT mice. Our data indicate that collagen XVIII may play an important role in corneal reinnervation after wounding.     

Attenuated response to liver injury in moesin-deficient mice: impaired stellate cell migration and decreased fibrosis

Hepatic stellate cells (HSCs) respond to injury with a coordinated set of events (termed activation), which includes migration and upregulation of matrix protein production. Cell migration requires an intact actin cytoskeleton that is linked to the plasma membrane by ezrin-radixin-moesin (ERM) proteins. We have previously found that the linker protein in HSCs is exclusively moesin. Here, we describe HSC migration and fibrogenesis in moesin-deficient mice. We developed an acute liver injury model that involved focal thermal denaturation and common bile duct ligation. HSC migration and collagen deposition were assessed by immunohistology and quantitative real-time PCR. Activated HSCs were isolated from wild-type or moesin-deficient mice for direct examination of migration. Activated HSCs from wild-type mice were positive for moesin. Migration of moesin-deficient HSCs was significantly reduced. In a culture assay, 22.1% of normal HSCs migrated across a filter in 36h. In contrast, only 1.3% of activated moesin-deficient HSCs migrated. Collagen deposition around the injury area similarly was reduced in moesin-deficient liver. The linker protein moesin is essential for HSC activation and migration in response to injury. Fibrogenesis is coupled to migration and reduced in moesin-deficient mice. Agents that target moesin may be beneficial for chronic progressive fibrosis.     

Effects of activated charcoal, explant size, explant position and sucrose concentration on plant and shoot regeneration of Lilium longiflorum via young stem culture

An efficient system for the in vitro plant and shootregeneration of Lilium longiflorum was developed andaccomplished using transverse thin cell layers (tTCL) of young stems.tTCLs were cut transversely along young stems from which the shoot-tipshad been removed. Sections were measured accurately using a graded gridand were cut in 4 mm × 4 mm × 1 mm cubes, eliminatingepidermal tissue, and were cultured on one-half MS medium containing 8 gl^–1 agar, different sucrose concentrations (10, 20, 30 or 40g l^–1), and with or without 1 mg l^–1 activatedcharcoal (AC). Plants formed on the surface of tTCLs within 60 days onone-half MS medium containing 8 g l^–1 agar and 20 gl^–1 sucrose. Sections of 1 mm taken just below the apicalarea developed buds within 15 days, whereas the sections closer to thebase required about 45 days. Shoot regeneration was enhanced whensucrose concentration was used at 30 or 40 g l^–1 after 60days of culture. No root formation occurred. Both shooting and rootingoccurred when sucrose was used at 20 g l^–1. The plantletswere transferred to soil and grew well under greenhouseconditions.     

A model cortical circuit for the storage of temporal sequences

Despite the fact that temporal information processing is of particular significance in biological memory systems, not much has yet been explored about how these systems manage to store temporal information involved in sequences of stimuli. A neural network model capable of learning and recalling temporal sequences is proposed, based on a neural mechanism in which the sequences are expanded into a series of periodic rectangular oscillations. Thus, the mathematical framework underlying the model, to some extent, is concerned with the Walsh function series. The oscillatory activities generated by the interplay between excitatory and inhibitory neuron pools are transmitted to another neuron pool whose role in learning and retrieval is to modify the rhythms and phases of the rectangular oscillations. Thus, a basic functional neural circuit involves three different neuron pools. The modifiability of rhythms and phases is incorporated into the model with the aim of improving the quality of the retrieval. Numerical simulations were conducted to show the characteristic features of the learning as well as the performance of the model in memory recall.     

A model of cortical memory processing based on columnar organization

Recent neurophysiological experiments using mammalian brains indicated that some cortical neurons exhibit oscillatory activities which can be of functional importance in visual perception. These findings suggest that the oscillation is an ubiquitous feature of cortical information processing carried out by columns which are receiving growing attention as functional subdivisions of cortical circuitry. On the assumption that a basic functional unit is a column comprising excitatory and inhibitory neurons, a network model of cortical memory processing which can account for these oscillations is proposed. Numerical simulations revealed that for appropriately determined parameters the network can attain memory-pattern retrieval resulting from fixed-point behaviour despite the fact that columns have the characteristic of oscillators. Received: 19 March 1993/Accepted in revised form: 23 September 1993     

Kuwanon W,a natural Diels—Alder type adduct from the root bark of Morus lhou

A natural Diels—Alder type adduct, named kuwanon W, was isolated from ethyl acetate extracts of the root bark of cultivated mulberry tree, and its structure was determined on the basis of spectral and chemical evidence. Kuwanon W is regarded biogenetically as a Diels—Alder adduct of a chalcone derivative and dehydroprenyl-flavone.