Transforming growth factor beta alters plasminogen activator activity in human skin fibroblasts

Adult human skin fibroblasts were used as a model to study the effects of transforming growth factor beta (TGF beta) on the secreted plasminogen activator (PA) activity of cultured cells. TGF beta, at nanogram concentrations, enhanced the secretion of pro-PA from two fibroblast strains in a time- and dose-dependent manner. The induced enzymatic activity was inhibited by anti-urokinase antibodies and it co-migrated with purified urokinase in polyacrylamide gels. The secretion of PA activity was abolished when cycloheximide (0.1 microgram/ml) was added to the cultures. The activity was thus dependent on protein synthesis rather than just on direct activation of a plasminogen proactivator. TGF beta had only a slight mitogenic effect on the test cells. Epidermal growth factor (EGF), platelet-derived growth factor (PDGF) and insulin were ineffective alone in inducing PA. Insulin, on the contrary, had an inhibitory effect on the TGF beta-induced PA activity. In addition to its effects on the secretion of PA, TGF beta enhanced the production of a proteinase inhibitor by these cells. The results suggest a role for TGF beta in the regulation of PA activity and pericellular proteolysis in fibroblastic cells.     

DNA barcodes identify Central Asian Colias butterflies (Lepidoptera,Pieridae)

A majority of the known Colias species (Lepidoptera: Pieridae, Coliadinae) occur in the mountainous regions of Central-Asia, vast areas that are hard to access, rendering the knowledge of many species limited due to the lack of extensive sampling. Two gene regions, the mitochondrial COI ‘barcode’ region and the nuclear ribosomal protein RpS2 gene region were used for exploring the utility of these DNA markers for species identification. A comprehensive sampling of COI barcodes for Central Asian Colias butterflies showed that the barcodes facilitated identification of most of the included species. Phylogenetic reconstruction based on parsimony and Neighbour-Joining recovered most species as monophyletic entities. For the RpS2 gene region species-specific sequences were registered for some of the included Colias spp. Nevertheless, this gene region was not deemed useful as additional molecular ‘barcode’. A parsimony analysis of the combined COI and RpS2 data did not support the current subgeneric classification based on morphological characteristics.     

Hepatocyte growth factor releases mink epithelial cells from transforming growth factor beta1-induced growth arrest by restoring Cdk6 expression and cyclin E-associated Cdk2 activity

Transforming growth factor beta (TGF-beta) potently suppresses Mv1Lu mink epithelial cell growth, whereas hepatocyte growth factor (HGF) counteracts TGF-beta-mediated growth inhibition and induces Mv1Lu cell proliferation (J. Taipale and J. Keski-Oja, J. Biol. Chem. 271:4342-4348, 1996). By addressing the cell cycle regulatory mechanisms involved in HGF-mediated release of Mv1Lu cells from TGF-beta inhibition, we show that increased DNA replication is accompanied by phosphorylation of the retinoblastoma protein and alternative regulation of cyclin-Cdk-inhibitor complexes. While TGF-beta treatment decreased the expression of Cdk6, this effect was counteracted by HGF, followed by partial restoration of cyclin D2-associated kinase activity. Notably, HGF failed to prevent TGF-beta induction of p15 and its association with Cdk6. However, HGF reversed the TGF-beta-mediated decrease in Cdk6-associated p27 and cyclin D2-associated Cdk6, suggesting that HGF modifies the TGF-beta response at the level of G1 cyclin complex formation. Counteraction of TGF-beta regulation of Cdk6 by HGF may in turn affect the association of p27 with Cdk2-cyclin E complexes. Though HGF did not differentially regulate the total levels of p27 in TGF-beta-treated cells, p27 immunodepletion experiments suggested that upon treatment with both growth factors, less p27 is associated with Cdk2-cyclin E complexes, in parallel with restoration of the active form of Cdk2 and the associated kinase activity. The results demonstrate that HGF intercepts TGF-beta cell cycle regulation at multiple points, affecting both G1 and G1-S cyclin kinase activities.     

Proteasomal activity modulates TGF-ss signaling in a gene-specific manner

Myosin heavy chain kinase A (MHCK A) modulates myosin II filament assembly in the amoeba Dictyostelium discoideum. MHCK A localization in vivo is dynamically regulated during chemotaxis, phagocytosis, and other polarized cell motility events, with preferential recruitment into anterior filamentous actin (F-actin)-rich structures. The current work reveals that an amino-terminal segment of MHCK A, previously identified as forming a coiled-coil, mediates anterior localization. MHCK A co-sediments with F-actin, and deletion of the amino-terminal domain eliminated actin binding. These results indicate that the anterior localization of MHCK A is mediated via direct binding to F-actin, and reveal the presence of an actin-binding function not previously detected by primary sequence evaluation of the coiled-coil domain.     

Variations in faecal bacterial enzyme activities and associations with bowel function and diet in elderly subjects

Daily and inter-individual variations of faecal bacterial β-glucuronidase and β-glucosidase activities and their associations with parameters of bowel function were studied in 10 residents of an old people's home during two 1-week periods 2 weeks apart. The effect of sampling method (a spot sample vs an aliquot of the homogenized sample from a total daily collection) on the activities of these enzymes and that of urease was also assessed. Intestinal transit time was determined using the radio-opaque Sitzmark^®; capsules, and questionnaires on bowel function and intakes of fluids and fibre-containing foods were completed. The mean (95% confidence interval) β-glucuronidase and β-glucosidase levels were 3·08 (2·75–3·41) and 11·53 (10·79–12·26) nmol min⁻¹ mg protein⁻¹. Daily variations in enzyme activities within individuals were not significant ( P = 0·277 and 0·990, respectively), whilst those between individuals were highly significant ( P = 0·000). Faecal frequency correlated negatively with β-glucuronidase and urease, but no other associations of the enzymic activities with parameters of bowel function and diet were observed. β-Glucuronidase and β-glucosidase were not affected by the sampling method, while significantly higher urease was obtained by spot sampling as compared with the aliquot representing the total daily collection. Large inter-individual variations in faecal enzyme activities should be taken into consideration when planning experiments and interpreting results on these faecal parameters.     

Light responses of mire mosses – a key to survival after water‐level drawdown?

Mosses are important ecosystem engineers in mires. Their existence may be threatened directly or indirectly by anthropogenic drying, which further leads to shading and changed competition conditions via increased arboreal plant cover. Yet, some species are able to acclimate to the changing habitat, while some give way to new colonizers. In the shaded conditions, acclimation or adaptation to low light levels is likely to be a winning strategy to survive. We studied the light responses of photosynthesis and photosynthetic pigment concentrations in mosses from an open mire and its shaded, i.e. drained and forested counterpart. Against our expectations, the Sphagnum species found only in the open habitat had lower photosynthetic capacity and maximum quantum yield than those found to grow in the shade. Chlorophyll fluorescence results suggested that photoinhibitory damage to photosystem II is responsible for the low photosynthetic performance of the Sphagna of the open habitat, which were inefficient to utilize any light level. In the shaded habitat, Sphagnum mosses showed adaptation to lower light conditions only by possessing a higher chlorophyll content. Pleurozium schreberi reached photosynthetic light saturation at half the irradiance level compared to Sphagna. The lack of efficient photoprotection or repair mechanism after photodamage may constrain the success of these species in the open habitat. Thus, the dominant Sphagna in the open pristine conditions seem to be stress tolerant, while the dominants of the shaded drained mire appear to be species capable of maximizing their growth and production to compete in the unstressful conditions in terms of light and desiccation.     

Differential epithelium DNA damage response to ATM and DNA-PK pathway inhibition in human prostate tissue culture

The ability of cells to respond and repair DNA damage is fundamental for the maintenance of genomic integrity. Ex vivo culturing of surgery-derived human tissues has provided a significant advancement to assess DNA damage response (DDR) in the context of normal cytoarchitecture in a non-proliferating tissue. Here, we assess the dependency of prostate epithelium DDR on ATM and DNA-PKcs, the major kinases responsible for damage detection and repair by nonhomologous end-joining (NHEJ), respectively. DNA damage was caused by ionizing radiation (IR) and cytotoxic drugs, cultured tissues were treated with ATM and DNA-PK inhibitors, and DDR was assessed by phosphorylation of ATM and its targets H2AX and KAP1, a heterochromatin binding protein. Phosphorylation of H2AX and KAP1 was fast, transient and fully dependent on ATM, but these responses were moderate in luminal cells. In contrast, DNA-PKcs was phosphorylated in both luminal and basal cells, suggesting that DNA-PK-dependent repair was also activated in the luminal cells despite the diminished H2AX and KAP1 responses. These results indicate that prostate epithelial cell types have constitutively dissimilar responses to DNA damage. We correlate the altered damage response to the differential chromatin state of the cells. These findings are relevant in understanding how the epithelium senses and responds to DNA damage.Key words: DNA damage, prostate, γH2AX, ATM, DNA-PK     

Melanoma cell lines are susceptible to histone deacetylase inhibitor TSA provoked cell cycle arrest and apoptosis

Melanoma is the most aggressive of skin cancers because of its high resistance to currently available therapy. Although melanoma cells often retain wild-type p53 tumour suppressor protein and express it at high levels, the p53 mediated apoptosis pathway is suppressed. Histone deacetylase (HDAC) inhibitors are a promising group of compounds inducing differentiation, growth arrest and apoptosis in tumour cells in preclinical studies. We have studied the cellular effects of trichostatin A (TSA), a HDAC inhibitor, in a panel of melanoma cell lines and its mechanism of action in relation to p53. TSA stabilized wild-type p53, but p53 protein accumulation was overridden by simultaneous downregulation of p53 mRNA leading to a decrease in p53 protein. While growth arrest was induced in all cell lines studied and apoptosis in most (6/7), these cellular effects were independent of the p53 status of the cells. Inhibiting p53 function by a dominant negative p53 (p53(175His)) confirmed that the HDAC inhibitor induced apoptosis was independent of wild-type p53, even though TSA slightly activated p53 in a reporter assay. The results indicate that while the action of TSA is independent of p53, the activation of the apoptosis pathway by the HDAC inhibitors may provide therapeutic approaches for melanoma treatment.