Activated Rac1 Selectively Up-regulates the Expression of Integrin α6β4 and Induces Cell Adhesion and Membrane Ruffles of Nonadherent Colon Cancer Colo201 Cells

Functions of small GTPases in integrin expression were investigated when the interaction of nonadherent human colon carcinoma 201 cells with the extracellular matrix (ECM) was examined. By transfection of the constitutively active form of a small GTPase Rac1, Rac V12, adhesion of cells to the ECM increased with concomitant cell spreading and formation of membrane ruffles. Activated Cdc42 and Cdc42 V12, but not wild-type Rac1, Cdc42, or RhoA, also induced the adhesion and spreading of Colo201 cells. This adhesion is integrin β4 dependent since an antibody for integrin β4 inhibited the RacV12-dependent cell adhesion and numbers of adhesive cells on laminin-coated plates exceeded those on collagen- and fibronectin-coated plates. By immunofluorescence, in addition to clustering of integrin molecules, expression of integrin α6β4 on the cell surface of Rac V12- and Cdc42 V12-expressing cells was selectively up-regulated without an increase in biosynthesis of α6β4 integrin. Treatment of Rac V12-expressing cells with wortmannin or LY294002, specific inhibitors of phosphoinositide 3-OH kinase, decreased the up-regulated α6β4 and cell adhesion. In light of this evidence, we propose that the regulation of integrin α6β4 expression induced by Rac1 and Cdc42 may play an important role in cell adhesion and tumorigenesis of colon carcinoma cells.     

Competition between two similar plant varieties,green shrunk perilla and red shrunk perilla,in mixed cultures

Influences of plant density and time after seeding on the growth of two horticultural forms of perilla (Perilla frutescens var.crispa), green shrunk perilla (f.viridi-crispa) and red shrunk perilla (f.crispa), were examined in a mixed culture experiment. Relationships between mean individual plant weight and plant density in mixed populations were approximated by Ogawa's non-interaction type (NI-type) reciprocal equation. The density conversion factors in the equation for green and red perillas were always, respectively, smaller and larger than unity, suggesting that effects of a green perilla on the other individuals were always stronger than those of a red one in a mixed population. All coefficients in the NI-type reciprocal equation were expressed as functions of time after seeding. As a result, time trends of mean individual plant weights for both species in mixed populations could be reasonably estimated for different plant densities and mixed proportions. The results were also applied to Lotka-Volterra's equation. Time trends of Lotka-Volterra's competition coefficients for both plants could be calculated and were compared with those of density conversion factors.     

Acetyl-CoA Carboxylase in Higher Plants: Most Plants Other Than Gramineae Have Both the Prokaryotic and the Eukaryotic Forms of This Enzyme

The presence and the absence of a prokaryote type and a eukaryotetype of acetyl-CoA carboxylase (EC 6.4.1.2 [EC] ; ACCase) were examinedin members of 28 plant families by two distinct methods: thedetection of biotinylated subunits of ACCase with a streptavidinprobe, and the detection of the accD gene, which encodes a subunitof the prokaryotic ACCase, by Southern hybridization analysis.The protein extracts of all the plants studied contained a biotinylatedpolypeptide of 220 kDa, which was probably the eukaryotic ACCase.All the plants but those belonging to Gramineae also containeda biotinylated polypeptide of ca. 35 kDa, which is a putativesubunit of the prokaryotic ACCase. In all plants but those inGramineae, the ca. 35 kDa polypeptide was found in the proteinextracts of plastids, while the 220 kDa polypeptide was absentfrom these plastid extracts. The plastid extracts of the plantsin Gramineae contained the 220 kDa polypeptide, as did the homogenatesof the leaves. Southern hybridization analysis demonstratedthat all the plants but those in the Gramineae contained theaccD gene. These findings suggest that most higher plants havethe prokaryotic ACCase in the plastids and the eukaryotic ACCasein the cytosol. Only Gramineae plants might contain the eukaryoticACCases both in the plastids and in the cytosol. The originof the plastid-located eukaryotic ACCase in Gramineae is discussedas the first possible example of substitution of a plastid geneby a nuclear gene for a non-ribosomal component. ⁴Present address: Plant-Growth Regulation Laboratory, The Instituteof Physical and Chemical Research (RIKEN), Hirosawa 2-1, Wako,351-01 Japan ⁵Present address: Laboratory of Plant Molecular Biology, Schoolof Agricultural Sciences, Nagoya University, Chikusa-ku, Nagoya,464-01 Japan     

Alkaloid of Stephania japonica Miers On Protostephanine (12)

By the reduction of protostephanine methylmethine, two kinds of methines (α- and β-isomer) were isolated, and a stable nitrogen-free substance was successfully obtained by its Hofmann degradation. This in connection with other evidence, makes possible a complete assignement of structure for protostephanine.     

The spread of Rice dwarf virus among cells of its insect vector exploits virus-induced tubular structures

Various cytopathological structures, known as inclusion bodies, are formed upon infection of cultured leafhopper cells by Rice dwarf virus, a member of the family Reoviridae. These structures include tubules of approximately 85 nm in diameter which are composed of the nonstructural viral protein Pns10 and contain viral particles. Such tubular structures were produced in heterologous non-host insect cells that expressed Pns10 of the virus. These tubules, when associated with actin-based filopodia, were able to protrude from the surface of cells and to penetrate neighboring cells. A binding assay in vitro revealed the specific binding of Pns10 to actin. Infection of clusters of cells was readily apparent 5 days after inoculation at a low multiplicity of infection with the virus, even in the presence of neutralizing antibodies. However, treatment of host cells with drugs that inhibited the elongation of actin filaments abolished the extension of Pns10 tubules from the surface of cells, with a significant simultaneous decrease in the extent of infection of neighboring cells. These results together revealed a previously undescribed aspect of the intercellular spread of Rice dwarf virus, wherein the virus exploits tubules composed of a nonstructural viral protein and actin-based filopodia to move into neighboring cells.     

Targeted ablation of osteocytes induces osteoporosis with defective mechanotransduction

Bone remodeling is performed by osteoclasts and osteoblasts at the bone surface. Inside of bone is a network of numerous osteocytes, whose specific function has remained an enigma. Here we describe a transgenic mouse model in which inducible and specific ablation of osteocytes is achieved in vivo through targeted expression of diphtheria toxin (DT) receptor. Following a single injection of DT, approximately 70%–80% of the osteocytes, but apparently no osteoblasts, were killed. Osteocyte-ablated mice exhibited fragile bone with intracortical porosity and microfractures, osteoblastic dysfunction, and trabecular bone loss with microstructural deterioration and adipose tissue proliferation in the marrow space, all of which are hallmarks of the aging skeleton. Strikingly, these “osteocyte-less” mice were resistant to unloading-induced bone loss, providing evidence for the role of osteocytes in mechanotransduction. Thus, osteocytes represent an attractive target for the development of diagnostics and therapeutics for bone diseases, such as osteoporosis.     

Effects of cryopreservation of mouse embryos and in vitro fertilization on genotypic frequencies in colonies

To evaluate the effects of cryopreservation and in vitro fertilization (IVF) on genotypic frequencies in mouse colonies, genotypic frequencies at 15 biochemical, 4 immunological and 20 microsatellite loci were examined in three colonies of MCH (ICR) mice derived from noncryopreserved embryos obtained by natural mating without the induction of superovulation, cryopreserved embryos obtained by natural mating with the induction of superovulation, and cryopreserved embryos obtained by the induction of superovulation and IVF. Three (Pgm-1, Ldr-1 and Hbb) out of the 15 biochemical loci, two (Thy-1 and H2K) out of four immunological loci and five (D5Mit18, D6Mit15, D12Mit5, D13Mit26, and D14Mit7) out of 20 microsatellite loci that showed polymorphisms in every colony were used for detection of genotypic frequencies. The genotypic frequencies of the loci in the three colonies did not differ from the predicted genotypic frequencies (P > 0.05). The results suggested that genetic drift does not occur among colonies established from treated and untreated embryos, and it was clear that the embryo banking by cryopreservation is suitable for preservation of outbred stock without genetic drift.     

Sleep-time variation for ethanol and the hypnotic drugs tribromoethanol, urethane, pentobarbital, and propofol within outbred ICR mice.

To evaluate the phenotypic variation within a commercial outbred mouse stock, we examined sleep-time (or duration of loss of righting reflex) of outbred ICR mice after i.p. injection of ethanol (4.0 g/kg of body weight), urethane (1.3 g), tribromoethanol (250 mg), and pentobarbital (60 mg), and after i.v. injection of propofol (30 mg). We observed high-grade individual differences in sleep-time that ranged from 0 to 179 min, 83.1 +/- 4.3 (mean and SEM of 100 mice) for ethanol; 0 to 169 min, 64.5 +/- 3.1 for pentobarbital; 0 to 160 min, 36.6 +/- 3.6 for urethane; 0 to 120 min, 21.5 +/- 2.2 for tribromoethanol; and 3 to 20.5 min, 7.1 +/- 0.3 for propofol. This extensive phenotypic variance within the outbred stock was as great as the variation reported among inbred strains or selected lines, and the varied susceptibility within the colony was inherited by Jcl:ICR-derived inbred strains IAI, ICT, IPI, and IQI. The range of sleep-time variance for ethanol, pentobarbital, urethane, tribromoethanol, and propofol within four-way cross hybrid Jcl:MCH(ICR) mice was 86.6%, 63.3%, 124%, 61.0%, and 53.1% that of outbred Jcl:ICR mice, respectively. The present study indicates that phenotypic variance within an outbred Jcl:ICR stock was at high risk for susceptibility to the drugs that depress the central nervous system and that Jcl:ICR-derived inbreds may be an excellent source of animal models for studying the anesthesia gene.     

Lack of B cell leakiness in BALB/cA-nu, scid double mutant mice.

BALB/cA mice homozygous for both nu and scid mutations (BALB/cA-nu/nu, scid/scid) were developed by mating between BALB/cA-scid and BALB/cA-nu. These mice have greater longevity than C.B-17-scid because no thymic lymphoma occurs in them unlike in the latter. C.B-17-scid is known to show the leaky phenomenon in which a few clones of functional T and B cells develop in aged C.B-17-scid. Unexpectedly, the leaky B cells and T cells were absent or suppressed in BALB/cA-nu, scid mice when cytokine expressions were determined by RT-PCR, lymphocyte phenotypes by flow cytometry and serum immunoglobulin levels by ELISA. These results indicate that B cell leakiness may be induced by leaked T cells. BALB/cA-nu, scid mice may be useful as a recipient in allo- and xeno-transplantation experiments because of the absence of both thymic lymphomas and leakiness, in addition to lack of hair.