Variant forms of a group I intron in nuclear small-subunit rRNA genes of the marine red alga Porphyra spiralis var. amplifolia

A group IC1 intron occurs in nuclear small-subunit (18S) ribosomal RNA (SSU rRNA) genes of the marine red alga Porphyra spiralis var. amplifolia. This intron occurs at the same position as the self- splicing group IC1 introns in nuclear SSU rDNAs of the fungus Pneumocystis carinii and in the green alga Chlorella ellipsoidea and shares sequence identity with the Pneumocystis carinii intron in domains L1, P1, P2, and L2, outside the conserved core. Three size variants, differing in amount of sequence in L1, exist and are differentially distributed in geographically distinct populations. Preliminary data suggest that the largest variant can self-splice in vitro. Short open reading frames are present but do not correspond to known genes. Repeated nucleotide motifs, reminiscent of duplicated target sites of transposons or Alu elements, are associated with the intron and with one of the variant forms of L1. Insertions are present in nuclear SSU rDNAs of several other Porphyra species and of the red alga Bangia atropurpurea; insertionless rDNA variants also occur in several Porphyra species. Our observations are most readily explained by intron mobility, although it remains unclear how transfer could have been mediated between genomes of organisms as ecologically diverse as marine red algae, freshwater green algae, and a mammalian-pathogenic fungus.      

DNA sequence of the region in the genome of herpes simplex virus type 1 containing the exonuclease gene and neighbouring genes.

We report the sequence of a 7800 base pair region of herpes simplex virus type 1 DNA, representing approximately 0.16 to 0.20 map units in the genome. This contains sequences transcribed into a leftward oriented set of five 3' coterminal mRNAs, together with two rightward transcribed flanking genes. One of the leftward genes encodes the virus's alkaline exonuclease, but the other gene products are uncharacterized. The amino acid sequence of one encoded protein suggested that it is a membrane embedded species. The DNA sequence is densely utilised, with two predicted out-of-frame overlaps of coding sequences, and probably six occurrences of promoter elements within coding sequences. Homologues of five of the genes were found for the distantly related Epstein-Barr virus, with a similar overall relative arrangement.     

A cyclic AMP dependent protein kinase in Dictyostelium discoideum

A cyclic AMP-dependent protein kinase was found to appear during the time course of development of $\text{Dictyostelium}\text{discoideum}$. No cyclic AMP dependency was observed at any stage of development in crude 110,000 X G soluble extracts. After partial purification, however, extracts from post-aggregation stages contained enzyme that was activated up to 6-fold by cyclic AMP, whereas protein kinase from earlier stages was not affected by cyclic AMP. Likewise, cyclic AMP binding activity increased from the aggregation to the slug stage of development. Approximately one-half of the total cyclic AMP binding activity co-purified with the cyclic AMP dependent protein kinase. The enzyme from $\text{Dictyostelium}$ showed similarities to mammalian protein kinases with respect to its kinetic properties but differed in its behavior on ion-exchange chromatography.     

Src in cancer: deregulation and consequences for cell behaviour

Considerable evidence now implicates elevated expression and/or activity of Src in cancer development. In cells, endogenous Src is switched from an inactive to an active state by a variety of mechanisms that simultaneously relieve constraints on the kinase and protein-interacting Src homology (SH) domains. As a result, Src is translocated to the cell periphery, often to sites of cell adhesion, where myristylation mediates attachment to the inner surface of the plasma membrane. From these peripheral sites, Src's catalytic activity initiates intracellular signal transduction pathways that influence cell growth and adhesion strength, the latter contributing to control of cell migration. De-regulation in cancer cells may therefore enhance tumour growth and/or stimulate migratory or invasive potential in cells that would normally be relatively non-motile. Evidence now exists to suggest that Src may also influence the life or death decisions that cells make during many biological processes. Thus, Src modulation in cancer cells can alter cell responses that are often perturbed in cancer. Consequently, there is optimism that drugs which inhibit Src's kinase activity, or the activity of its downstream effectors, might have profound effects on cancer cell behaviour and be useful therapeutic agents.     

AvrBsT Acetylates Arabidopsis ACIP1, a Protein that Associates with Microtubules and Is Required for Immunity

Bacterial pathogens of plant and animals share a homologous group of virulence factors, referred to as the YopJ effector family, which are translocated by the type III secretion (T3S) system into host cells during infection. Recent work indicates that some of these effectors encode acetyltransferases that suppress host immunity. The YopJ-like protein AvrBsT is known to activate effector-triggered immunity (ETI) in Arabidopsis thaliana Pi-0 plants; however, the nature of its enzymatic activity and host target(s) has remained elusive. Here we report that AvrBsT possesses acetyltransferase activity and acetylates ACIP1 (for ACETYLATED INTERACTING PROTEIN1), an unknown protein from Arabidopsis. Genetic studies revealed that Arabidopsis ACIP family members are required for both pathogen-associated molecular pattern (PAMP)-triggered immunity and AvrBsT-triggered ETI during Pseudomonas syringae pathovar tomato DC3000 (Pst DC3000) infection. Microscopy studies revealed that ACIP1 is associated with punctae on the cell cortex and some of these punctae co-localize with microtubules. These structures were dramatically altered during infection. Pst DC3000 or Pst DC3000 AvrRpt2 infection triggered the formation of numerous, small ACIP1 punctae and rods. By contrast, Pst DC3000 AvrBsT infection primarily triggered the formation of large GFP-ACIP1 aggregates, in an acetyltransferase-dependent manner. Our data reveal that members of the ACIP family are new components of the defense machinery required for anti-bacterial immunity. They also suggest that AvrBsT-dependent acetylation in planta alters ACIP1''s defense function, which is linked to the activation of ETI.     

Interleukin-23 is critical for full-blown expression of a non-autoimmune destructive arthritis and regulates interleukin-17A and RORγt in γδ T cells

Introduction

A protein analysis using a mass spectrometry indicated that there are serum proteins showing significant quantitative changes after the administration of infliximab. Among them, connective tissue growth factor (CTGF) seems to be related to the pathogenesis of rheumatoid arthritis (RA). Therefore, this study was conducted to investigate how CTGF is associated with the disease progression of RA.

Methods

Serum samples were collected from RA patients in active or inactive disease stages, and before or after treatments with infliximab. CTGF production was evaluated by ELISA, RT-PCR, indirect immunofluorescence microscopy, and immunoblotting. Osteoclastogenesis was evaluated using tartrate-resistant acid phosphatase (TRAP) staining, a bone resorption assay and osteoclasts specific catalytic enzymes productions.

Results

The serum concentrations of CTGF in RA were greater than in normal healthy controls and disease controls. Interestingly, those were significantly higher in active RA patients compared to inactive RA patients. Furthermore, the CTGF levels significantly were decreased by infliximab concomitant with the disease amelioration. In addition, tumour necrosis factor (TNF)α can induce the CTGF production from synovial fibroblasts even though TNFα can oppositely inhibit the production of CTGF from chondrocytes. CTGF promoted the induction of the quantitative and qualitative activities of osteoclasts in combination with M-CSF and receptor activator of NF-κB ligand (RANKL). In addition, we newly found integrin αVβ3 on the osteoclasts as a CTGF receptor.

Conclusions

These results indicate that aberrant CTGF production induced by TNFα plays a central role for the abnormal osteoclastic activation in RA patients. Restoration of aberrant CTGF production may contribute to the inhibition of articular destruction in infliximab treatment.     

mDia1 Targets v-Src to the Cell Periphery and Facilitates Cell Transformation,Tumorigenesis, and Invasion

The small GTPase Rho regulates cell morphogenesis through remodeling of the actin cytoskeleton. While Rho is overexpressed in many clinical cancers, the role of Rho signaling in oncogenesis remains unknown. mDia1 is a Rho effector producing straight actin filaments. Here we transduced mouse embryonic fibroblasts from mDia1-deficient mice with temperature-sensitive v-Src and examined the involvement and mechanism of the Rho-mDia1 pathway in Src-induced oncogenesis. We showed that in v-Src-transduced mDia1-deficient cells, formation of actin filaments is suppressed, and v-Src in the perinuclear region does not move to focal adhesions upon a temperature shift. Consequently, membrane translocation of v-Src, v-Src-induced morphological transformation, and podosome formation are all suppressed in mDia1-deficient cells with impaired tyrosine phosphorylation. mDia1-deficient cells show reduced transformation in vitro as examined by focus formation and colony formation in soft agar and exhibit suppressed tumorigenesis and invasion when implanted in nude mice in vivo. Given overexpression of c-Src in various cancers, these findings suggest that Rho-mDia1 signaling facilitates malignant transformation and invasion by manipulating the actin cytoskeleton and targeting Src to the cell periphery.The small GTPase Rho functions as a molecular switch in cell morphogenesis through remodeling of the actin cytoskeleton (3, 14). Rho cycles between the inactive GDP-bound form and the active GTP-bound form. This process is controlled by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) specific to Rho; the former group catalyzes the exchange of GDP to GTP (50), and the latter accelerates the hydrolysis of bound GTP (24). When Rho is activated in fibroblasts, actin stress fibers are formed. Rho proteins are frequently overexpressed in human cancers, such as cancers of the colon and breast and lung and testicular germ cell tumors (34). A positive correlation between the expression level of RhoA and disease progression was also reported in breast cancer and testicular germ cell tumors. RhoC, on the other hand, has been repeatedly identified as a gene positively associated with metastasis (4, 21, 40). The clinical significance of Rho in cancer is further implicated by a discovery that a RhoA GAP named Dlc-1 (deleted liver cancer 1) functions as a tumor suppressor in humans (47). Thus, it was known for some time that heterozygous deletions on chromosome 8p22 are common in human tumors, such as cancers of the breast, prostate, lung (5, 22), and especially liver (15). Recent studies have revealed a strong association of deletion of DLC-1 in this region with clinical cancers, and complementary in vitro experiments showed that DLC-1 functions as a potent tumor suppressor, depletion of which causes RhoA hyperactivation and results in tumorigenesis in harmony with other oncogenes, such as Myc and Ras (47). Importantly, heterozygous deletions in chromosome 8p22 are found to be nearly as common as that of TP53 in clinical cancers, indicating the significant importance of DLC-1 and Rho signaling in clinical tumors (18). Consistent with these findings, there are several reports on the requirement of Rho activity in cell transformation in vitro. For example, coexpression of Raf and dominant active RhoA facilitates focus formation, and expression of dominant-negative RhoA suppresses oncogenic Ras-induced focus formation in NIH 3T3 cells (30). In addition, active forms of Rho GEFs, such as Dbl and Ect2, have potent transforming activities in cultured cells in vitro (31). Thus, there are ample in vitro and clinical data indicating the involvement of Rho signaling in oncogenesis.Cell transformation often leads to a change in cell morphology. This morphological change associates with a change in the organization of actin filaments. Nontransformed cells often have thick bundled actin fibers known as stress fibers. When transformed by some oncogenes, such as Ras and v-Src, the actin stress fibers disappear and the cells dramatically alter their shape to the round refractile cell body (49). Alternatively, actin dot structures called podosomes are often formed. This remodeling of the cytoskeleton is believed to contribute to several aspects of the transformed phenotype, including adhesion-independent cell growth and increased migration abilities. Such actin remodeling associated with oncogenesis appears at odds with the requirement of Rho signaling in oncogenesis, because Rho activation leads to formation of actin fibers. Thus, there is a paradox of why transformed cells require Rho signaling yet show dissolution of actin cytoskeleton (27).Among many Rho effectors, two effector molecules, named mDia (44) and ROCK (11), have important roles in actin cytoskeleton remodeling (27). mDia produces straight actin filaments by catalyzing actin nucleation and polymerization, and ROCK activates myosin to cross-link actin filaments for induction of actomyosin bundles and contractility. Further, mDia is potentially linked to Rac activation and membrane ruffle formation through c-Src-induced phosphorylation of focal adhesion proteins, and ROCK antagonizes this mDia action (42). Thus, actin remodeling inside the cell can be determined primarily by the balance between mDia and ROCK activities. Of the two, the involvement of ROCK in tumors has been widely examined by the use of its small molecule inhibitors, such as Y-27632 (26, 43), and the Rho-ROCK pathway has been strongly implicated in cancer migration and tumor metastasis and invasion. On the other hand, the role of ROCK in oncogenesis remains ambiguous. While its requirement in Ras-induced cell transformation was indicated by the use of Y-27632, examination in Ras-transformed cells revealed that the majority of ROCK is sequestered in an inactive pool by sustained extracellular signal regulated-kinase (ERK)-mitogen-activated protein (MAP) kinase activity under active Ras (33), which might be one of the mechanisms for dissolution of stress fibers found in Ras transformants. Thus, how Rho signaling contributes to oncogenesis remains an open question.Study of Rho effectors other than ROCK has been hampered by the absence of available inhibitors. Recently we generated mDia1 knockout mice (36). Here, we used mouse embryonic fibroblast (MEF) cells derived from these mice and analyzed the involvement of mDia1 and its mechanism of action in v-Src-induced cell transformation and tumorigenesis. v-Src is the oldest widely studied oncogene, yet it remains unknown where in the cell it exerts its oncogenic potential. It was previously reported that temperature-sensitive (ts) v-Src accumulates in the perinuclear region at the restrictive temperature and migrates to the periphery upon a temperature shift in a manner dependent on the actin cytoskeleton and Rho (6, 37). However, the underlying mechanism of this v-Src targeting has not been fully elucidated, and whether this targeting is required for v-Src-induced oncogenesis remains to be shown. Using mDia1-deficient MEF cells, we have addressed these questions. Here we have shown that actin filaments produced by mDia1 are a prerequisite for v-Src targeting, and this v-Src targeting is critical for its role in cell transformation and tumorigenesis. Our results further show that the Rho-mDia1 pathway functions as a link between oncogenesis and invasion.