The matrix metalloproteinase 9 (mmp-9) hemopexin domain is a novel gelatin binding domain and acts as an antagonist

Matrix metalloproteinases (MMPs) are involved in the remodeling processes of the extracellular matrix and the basement membrane. Most MMPs are composed of a regulatory, a catalytic, and a hemopexin subunit. In many tumors the expression of MMP-9 correlates with local tumor growth, invasion, and metastasis. To analyze the role of the hemopexin domain in these processes, the MMP-9 hemopexin domain (MMP-9-PEX) was expressed as a glutathione S-transferase fusion protein in Escherichia coli. After proteolytic cleavage, the isolated PEX domain was purified by size exclusion chromatography. In a zymography assay, MMP-9-PEX was able to inhibit MMP-9 activity. The association and dissociation rates for the interaction of MMP-9-PEX with gelatin were determined by plasmon resonance. From the measured rate constants, the dissociation constant was calculated to be K(d) = 2,4 x 10(-8) m, demonstrating a high affinity between MMP-9-PEX and gelatin. In Boyden chamber experiments the recombinant MMP-9-PEX was able to inhibit the invasion of melanoma cells secreting high amounts of MMP-9 in a dose-dependent manner. These data demonstrate for the first time that the hemopexin domain of MMP-9 has a high affinity binding site for gelatin, and the particular recombinant domain is able to block MMP-9 activity and tumor cell invasion. Because MMP-9 plays an important role in metastasis, this antagonistic effect may be utilized to design MMP inhibition-based cancer therapy.     

The origin of a novel form of Senecio (Asteraceae) restricted to sand dunes in southern Sicily

The taxonomy of diploid Mediterranean Senecio sect. Senecio (Asteraceae) is complex, owing to a recent species radiation, high morphological plasticity and occasional interspecific hybridization. A study was conducted to resolve the origin of a novel form of Senecio restricted to sand dunes in southern Sicily, Italy. This has been described previously as morphologically intermediate to Senecio gallicus and Senecio glaucus ssp. coronopifolius, indicating a possible hybrid origin, or as a variant of Senecio leucanthemifolius. Plants of this form grown in a glasshouse were morphologically intermediate to S. glaucus and S. leucanthemifolius, but were also similar to some cultivated individuals of S. gallicus. No evidence for a hybrid origin was obtained from a survey of random amplified polymorphic DNA variation; instead the plants surveyed were most closely allied to Tunisian S. glaucus. They were also polymorphic for the same set of cpDNA haplotypes present in Tunisian S. glaucus. We conclude that the Sicilian Senecio is a variant form of North African S. glaucus ssp. coronopifolius, which most probably dispersed to sand dunes in southern Sicily in the relatively recent past. The presence of several cpDNA haplotypes in this material indicates that there have been multiple introductions of the species to Sicily.     

Body elongation and decreased reproductive output within a restricted clade of lizards (Reptilia: Scincidae)

Relationships between body shape and relative abdominal size were compared among differentially elongate species within the scincid lizard genus Brachymeles , to investigate how morphological evolution affects the proportion of body volume available to hold eggs and offspring. Relative abdominal size is inversely related to elongation, suggesting that relative clutch mass decreases with addition of abdominal body segments. Shape-volume relationships contradict trends seen in comparisons among distantly related limbed and limbless squamates (lizards and snakes), in which snakes have relatively more abdominal volume. Comparison within a phylogenetically restricted group allows the identification of functional and ontogenetic factors potentially limiting reproductive output. In Bruchymeles , constraining factors include retention of anterior body segments bearing parasternal ribs, which prevents extension of the clutch anteriorly within the body, and reduction of allometry of abdominal segments, which provides extended series of uniformly-sized vertebrae for limbless locomotion, but reduces the relative size of the abdomen. The latter trait is associated with overall size reduction, which affects relative egg-size and packing. Factors constraining abdominal volume in this genus are probably common to other elongate lizards, a morphological group that has been rarely represented in comparative studies of life history.     

The anti-fibrotic effect of liver growth factor is associated with decreased intrahepatic levels of matrix metalloproteinases 2 and 9 and transforming growth factor beta 1 in bile duct-ligated rats

Liver growth factor (LGF), a mitogen for liver cells, behaves as an anti-fibrotic agent even in extrahepatic sites, but its mechanistic basis is unknown. We aimed to determine the intrahepatic expression pattern of key modulators of liver fibrosis in bile duct-ligated rats (BDL) after injection of LGF. BDL rats received either LGF (4.5 microg/ratXdose, two doses/week, at time 0 or 2 or 5w after operation, depending on the group (BDL+LGF groups, n=20) or saline (BDL+S groups, n=20). Groups were compared in terms of fibrosis (histomorphometry), liver function (aminopyrine breath test), matrix metalloproteinases MMP-2 and MMP-9, transforming growth factor beta 1 (TGF-beta1) and liver endoglin content (Western blotting), and serum tissue inhibitor of metalloproteinases 1 (TIMP-1) levels (ELISA). In BDL+LGF rats, the fibrotic index was significantly lower at 5w, p=0.006, and at 8w, p=0.04, than in BDL+S rats. Liver function values in BDL+LGF rats were higher than those obtained in BDL+S rats (80% at 5w and 79% at 8w, versus 38% and 29%, p<0.01, taking healthy controls as 100%). Notably, in BDL+LGF rats the intrahepatic expression levels of both MMPs were lower at 2w (MMP-2, p=0.03; MMP-9, p=0.05) and 5w (MMP-2, p=0.05, MMP-9, p=0.04). In addition, the hepatic TGF-beta1 level in BDL+LGF rats was lower at 2w (36%, p=0.008), 5w (50%) and 8wk (37%), whereas intrahepatic endoglin expression remained constant in all BDL rats studied. LGF ameliorates liver fibrosis and improves liver function in BDL rats. The LGF-induced anti-fibrotic effect is associated with a decreased hepatic level of MMP-2, MMP-9 and TGF-beta1 in fibrotic rats.     

N-Myc downstream-regulated gene 2 (NDRG2) is a negative regulator of cell growth and its expression is decreased in cancers

Using subtraction cloning, we identified the human N-Myc Downstream- Regulated Gene-2 (hNDRG2), located at 14q11.2, as a candidate tumor suppressor gene. Semi-quantitative RT-PCR showed that the expression of hNDRG2 in 15 of 27 (56%) human GBM tissues and all 6 human glioblastoma cell lines was significantly lower than that in the normal brain. The expression of hNDRG2 also was evaluated in 60 lung-carcinoma patients. 17 of 26 cases of squamous carcinoma and 4 of 11 cases of small cell lung cancer displayed     

A novel protein phosphatase 2C family member (PP2Czeta) is able to associate with ubiquitin conjugating enzyme 9

In this study we have cloned a novel member of mouse protein phosphatase 2C family, PP2Czeta, which is composed of 507 amino acids and has a unique N-terminal region. The overall similarity of the amino acid sequence between PP2Czeta and PP2Calpha was 22%. On Northern blot analysis PP2Czeta was found to be expressed specifically in the testicular germ cells. PP2Czeta expressed in COS7 cells was able to associate with ubiquitin conjugating enzyme 9 (UBC9) and the association was enhanced by co-expression of small ubiquitin-related modifier-1 (SUMO-1), suggesting that PP2Czeta exhibits its specific role through its SUMO-induced recruitment to UBC9.     

The 'antimutagenic' effect of cinnamaldehyde is due to a transient growth inhibition

Addition of cinnamaldehyde to the selective medium causes a reduction in the number of revertant colonies of S. typhimurium or E. coli when the cells have been mutagenized with 4NQO but not when they have been mutagenized with MNNG. Toxicity of the cinnamaldehyde exposure depends largely on the status of growth and/or nutrient supply of the cells. We present evidence that simple growth inhibition due to lack of nutrients mimics the effect of cinnamaldehyde in 4NQO- and MNNG-treated cells. This argues that the reduction of mutant colonies is due to a transient growth retardation caused by cinnamaldehyde exposure, which presumably allows the cells to repair 4NQO-induced damage--but not MNNG-induced damage--via a more error-free pathway.     

Zidovudine (AZT) resistance in H9 cells due to decreased TK expression is associated with hypermethylation of TK gene

AZT resistant human T-lymphoid H9 cells, deficient in TK gene expression, re-expressed TK mRNA and regained the ability to metabolize AZT by exposure to the demethylation agent azacytidine (AzaCd). Cytotoxic and anti-HIV-1 effects of AZT were increased in H9 AZT resistant cells treated with AzaCd when compared to untreated cells. This leads to the assumption that drug induced DNA hypermethylation was involved in the TK gene-silencing mechanism. Our results suggest approaches using modulation of gene methylation for increasing antiviral efficiency of drugs.     

Platelet-derived growth factor C (PDGF-C), a novel growth factor that binds to PDGF alpha and beta receptor

We have characterized platelet-derived growth factor (PDGF) C, a novel growth factor belonging to the PDGF family. PDGF-C is a multidomain protein with the N-terminal region homologous to the extracellular CUB domain of neuropilin-1, and the C-terminal region consists of a growth factor domain (GFD) with homology to vascular endothelial growth factor (25%) and PDGF A-chain (23%). A serum-sensitive cleavage site between the two domains allows release of the GFD from the CUB domain. Competition binding and immunoprecipitation studies on cells bearing both PDGF alpha and beta receptors reveal a high affinity binding of recombinant GFD (PDGF-CC) to PDGF receptor-alpha homodimers and PDGF receptor-alpha/beta heterodimers. PDGF-CC exhibits greater mitogenic potency than PDGF-AA and comparable or greater mitogenic activity than PDGF-AB and PDGF-BB on several mesenchymal cell types. Analysis of PDGF-CC in vivo in a diabetic mouse model of delayed wound healing showed that PDGF-CC significantly enhanced repair of a full-thickness skin excision. Together, these studies describe a third member of the PDGF family (PDGF-C) as a potent mitogen for cells of mesenchymal origin in in vitro and in vivo systems with a binding pattern similar to PDGF-AB.     

The quest for microbial reductive dechlorination of C (2) to C (4) chloroalkanes is warranted

C (2) to C (4) chloroalkanes have been used for a wide range of industrial applications. Consequently, numerous leaks to the environment have occurred. It is generally observed that the lower chlorinated members of the group, containing 1-3 chlorine atoms, accumulate in environments where reductive conditions prevail. Their half-lives under these conditions often exceed several decades. To date, successes in rapid and complete in situ reductive dechlorination have only been obtained with tetrachloroethene (PCE) and trichloroethene (TCE), but not with chloroalkanes. Since the key-player PCE- and TCE-dechlorinating bacteria involved have been studied, these organisms could be used as very efficient tools for low-cost in situ bioremediation. Except for one 1,2-dichloroethane-dehalorespiring bacterium with limited application possibilities and a recent isolate which partly dechlorinates some polychloroethanes, all bacterial reductive conversions of C (2) to C (4) chloroalkanes are based on slow, mostly incomplete and poorly controllable cometabolic dechlorinations. Furthermore, metals such as Fe(0) cannot dechlorinate most lower-chlorinated C (2) to C (4) alkanes. Hence, pump and treat, or aerobic degradation are the applied technologies, although they are expensive and time-intensive. However, energetic consideration of chloroalkane dechlorination suggests that metabolizing anaerobes may exist. Isolation and characterization of these organisms is warranted in order to develop cost-efficient, controlled, fast and complete in situ remediation technologies.     

APLF (C2orf13) is a novel component of poly(ADP-ribose) signaling in mammalian cells

APLF is a novel protein of unknown function that accumulates at sites of chromosomal DNA strand breakage via forkhead-associated (FHA) domain-mediated interactions with XRCC1 and XRCC4. APLF can also accumulate at sites of chromosomal DNA strand breaks independently of the FHA domain via an unidentified mechanism that requires a highly conserved C-terminal tandem zinc finger domain. Here, we show that the zinc finger domain binds tightly to poly(ADP-ribose), a polymeric posttranslational modification synthesized transiently at sites of chromosomal damage to accelerate DNA strand break repair reactions. Protein poly(ADP-ribosyl)ation is tightly regulated and defects in either its synthesis or degradation slow global rates of chromosomal single-strand break repair. Interestingly, APLF negatively affects poly(ADP-ribosyl)ation in vitro, and this activity is dependent on its capacity to bind the polymer. In addition, transient overexpression in human A549 cells of full-length APLF or a C-terminal fragment encoding the tandem zinc finger domain greatly suppresses the appearance of poly(ADP-ribose), in a zinc finger-dependent manner. We conclude that APLF can accumulate at sites of chromosomal damage via zinc finger-mediated binding to poly(ADP-ribose) and is a novel component of poly(ADP-ribose) signaling in mammalian cells.     

Human Xip1 (C2orf13) is a novel regulator of cellular responses to DNA strand breaks

DNA strand breaks arise continuously as the result of intracellular metabolism and in response to a multitude of genotoxic agents. To overcome such challenges to genomic stability, cells have evolved genome surveillance pathways that detect and repair damaged DNA in a coordinated fashion. Here we identify the previously uncharacterized human protein Xip1 (C2orf13) as a novel component of the checkpoint response to DNA strand breaks. Green fluorescent protein-tagged Xip1 was rapidly recruited to sites of DNA breaks, and this accumulation was dependent on a novel type of zinc finger motif located in the C terminus of Xip1. The initial recruitment kinetics of Xip1 closely paralleled that of XRCC1, a central organizer of single strand break (SSB) repair, and its accumulation was both delayed and sustained when the detection of SSBs was abrogated by inhibition of PARP-1. Xip1 and XRCC1 stably interacted through recognition of CK2 phosphorylation sites in XRCC1 by the Forkhead-associated (FHA) domain of Xip1, and XRCC1 was required to maintain steady-state levels of Xip1. Moreover, Xip1 was phosphorylated on Ser-116 by ataxia telangiectasia-mutated in response to ionizing radiation, further underscoring the potential importance of Xip1 in the DNA damage response. Finally, depletion of Xip1 significantly decreased the clonogenic survival of cells exposed to DNA SSB- or double strand break-inducing agents. Collectively, these findings implicate Xip1 as a new regulator of genome maintenance pathways, which may function to organize DNA strand break repair complexes at sites of DNA damage.     

The anti-inflammatory effect of neuropeptide Y (NPY) in rats is dependent on dipeptidyl peptidase 4 (DP4) activity and age

Neuropeptide Y (NPY)-induced modulation of the immune and inflammatory responses is regulated by tissue-specific expression of different receptor subtypes (Y1–Y6) and the activity of the enzyme dipeptidyl peptidase 4 (DP4, CD26) which terminates the action of NPY on Y1 receptor subtype. The present study investigated the age-dependent effect of NPY on inflammatory paw edema and macrophage nitric oxide production in Dark Agouti rats exhibiting a high-plasma DP4 activity, as acknowledged earlier. The results showed that NPY suppressed paw edema in adult and aged, but not in young rats. Furthermore, plasma DP4 activity decreased, while macrophage DP4 activity, as well as macrophage CD26 expression increased with aging. The use of NPY-related peptides and Y receptor-specific antagonists revealed that anti-inflammatory effect of NPY is mediated via Y1 and Y5 receptors. NPY-induced suppression of paw edema in young rats following inhibition of DP4 additionally emphasized the role for Y1 receptor in the anti-inflammatory action of NPY. In contrast to the in vivo situation, NPY stimulated macrophage nitric oxide production in vitro only in young rats, and this effect was mediated via Y1 and Y2 receptors. It can be concluded that age-dependant modulation of inflammatory reactions by NPY is determined by plasma, but not macrophage DP4 activity at different ages.     

The Drosophila poly(A) binding protein-interacting protein, dPaip2, is a novel effector of cell growth

The 3' poly(A) tail of eukaryotic mRNAs and the poly(A) binding protein (PABP) play important roles in the regulation of translation. Recently, a human PABP-interacting protein, Paip2, which disrupts the PABP-poly(A) interaction and consequently inhibits translation, was described. To gain insight into the biological role of Paip2, we studied the Drosophila melanogaster Paip2 (dPaip2). dPaip2 is the bona fide human Paip2 homologue, as it interacts with dPABP, inhibits binding of dPABP to the mRNA poly(A) tail, and reduces translation of a reporter mRNA by approximately 80% in an S2 cell-free translation extract. Ectopic overexpression of dPaip2 in Drosophila wings and wing discs results in a size reduction phenotype, which is due to a decrease in cell number. Clones of cells overexpressing dPaip2 in wing discs also contain fewer cells than controls. This phenotype can be explained by a primary effect on cell growth. Indeed, overexpression of dPaip2 in postreplicative tissues inhibits growth, inasmuch as it reduces ommatidia size in eyes and cell size in the larval fat body. We conclude that dPaip2 inhibits cell growth primarily by inhibiting protein synthesis.