MMP-13 Regulates Growth of Wound Granulation Tissue and Modulates Gene Expression Signatures Involved in Inflammation, Proteolysis, and Cell Viability

Proteinases play a pivotal role in wound healing by regulating cell-matrix interactions and availability of bioactive molecules. The role of matrix metalloproteinase-13 (MMP-13) in granulation tissue growth was studied in subcutaneously implanted viscose cellulose sponge in MMP-13 knockout (Mmp13(-/-)) and wild type (WT) mice. The tissue samples were harvested at time points day 7, 14 and 21 and subjected to histological analysis and gene expression profiling. Granulation tissue growth was significantly reduced (42%) at day 21 in Mmp13(-/-) mice. Granulation tissue in Mmp13(-/-) mice showed delayed organization of myofibroblasts, increased microvascular density at day 14, and virtual absence of large vessels at day 21. Gene expression profiling identified differentially expressed genes in Mmp13(-/-) mouse granulation tissue involved in biological functions including inflammatory response, angiogenesis, cellular movement, cellular growth and proliferation and proteolysis. Among genes linked to angiogenesis, Adamts4 and Npy were significantly upregulated in early granulation tissue in Mmp13(-/-) mice, and a set of genes involved in leukocyte motility including Il6 were systematically downregulated at day 14. The expression of Pdgfd was downregulated in Mmp13(-/-) granulation tissue in all time points. The expression of matrix metalloproteinases Mmp2, Mmp3, Mmp9 was also significantly downregulated in granulation tissue of Mmp13(-/-) mice compared to WT mice. Mmp13(-/-) mouse skin fibroblasts displayed altered cell morphology and impaired ability to contract collagen gel and decreased production of MMP-2. These results provide evidence for an important role for MMP-13 in wound healing by coordinating cellular activities important in the growth and maturation of granulation tissue, including myofibroblast function, inflammation, angiogenesis, and proteolysis.     

The structure of the glial cell line-derived neurotrophic factor-coreceptor complex: insights into RET signaling and heparin binding

Glial cell line-derived neurotrophic factor (GDNF), a neuronal survival factor, binds its co-receptor GDNF family receptor alpha1 (GFR alpha 1) in a 2:2 ratio and signals through the receptor tyrosine kinase RET. We have solved the GDNF(2).GFR alpha 1(2) complex structure at 2.35 A resolution in the presence of a heparin mimic, sucrose octasulfate. The structure of our GDNF(2).GFR alpha 1(2) complex and the previously published artemin(2).GFR alpha 3(2) complex are unlike in three ways. First, we have experimentally identified residues that differ in the ligand-GFR alpha interface between the two structures, in particular ones that buttress the key conserved Arg(GFR alpha)-Glu(ligand)-Arg(GFR alpha) interaction. Second, the flexible GDNF ligand "finger" loops fit differently into the GFR alphas, which are rigid. Third, and we believe most importantly, the quaternary structure of the two tetramers is dissimilar, because the angle between the two GDNF monomers is different. This suggests that the RET-RET interaction differs in different ligand(2)-co-receptor(2)-RET(2) heterohexamer complexes. Consistent with this, we showed that GDNF(2).GFR alpha1(2) and artemin(2).GFR alpha 3(2) signal differently in a mitogen-activated protein kinase assay. Furthermore, we have shown by mutagenesis and enzyme-linked immunosorbent assays of RET phosphorylation that RET probably interacts with GFR alpha 1 residues Arg-190, Lys-194, Arg-197, Gln-198, Lys-202, Arg-257, Arg-259, Glu-323, and Asp-324 upon both domains 2 and 3. Interestingly, in our structure, sucrose octasulfate also binds to the Arg(190)-Lys(202) region in GFR alpha 1 domain 2. This may explain how GDNF.GFR alpha 1 can mediate cell adhesion and how heparin might inhibit GDNF signaling through RET.     

Population genetic structure in the Temminck’s stint <Emphasis Type="Italic">Calidris temminckii</Emphasis>, with an emphasis on Fennoscandian populations

Temminck’s stint breeds in Eurasian arctic tundra and subarctic and temperate boreal zones in a range extending from Fennoscandia to easternmost Siberia. In contrast to the favourable global conservation status of the species, it has been classified as vulnerable in Finland and near threatened in Sweden. A fragment of the control region of mtDNA was sequenced from 127 individuals from breeding areas in Fennoscandia in the west (three populations) and in the eastern end of the range. The mtDNA variability and structuring were among the lowest values reported for waders (F _ST −0.02616). The mtDNA sequences revealed seven haplotypes, of which four were present in single individuals. The most common haplotype occurred in 81% of all individuals and in all birds in the Siberian sample. There was evidence of two maternal lineages. The most common lineage occurred in 95% of the individuals and was the only one present in the Siberian sample. The lineages coexisted in all three Fennoscandian populations, indicating a secondary contact of two previously isolated populations. The mtDNA variation and the mitochondrial nucleotide and haplotype diversities indicated panmixis of the populations. However, a higher degree of population differentiation was detected in microsatellite allele frequencies (125 birds, six loci) in Fennoscandia between the Bothnian Bay population and the two inland populations (Lapland and southern Norway). The difference may be caused by the female-biased dispersal pattern of the species. In addition, the Bothnian Bay population appeared to be genetically bottlenecked, an observation in concordance with the recent decimation of the population.     

Restored cut-away peatland as a sink for atmospheric CO2

In a field study, we examined the relationship between vegetation, abiotic factors and the CO₂ exchange dynamics of a cut-away peatland 20 years after production had ended. The main objective was to determine the effect of rewetting on the CO₂ exchange dynamics, measured separately in Eriophorum vaginatum tussocks and intertussocks (almost non-vegetated surfaces) using closed-chamber techniques, one growing season before and three growing seasons after the rewetting treatment. Rewetting lowered total respiration (R _TOT) and increased gross photosynthesis (P _G), which resulted in a higher incorporation of CO₂ into the system. The seasonal CO₂ balance for the almost continuously submerged section of the rewetted site became positive 2 years after rewetting (9.1 g CO₂-C m⁻²), and it was still higher in the 3rd year (64.5 g CO₂-C m⁻²), i.e. the system accumulated carbon. In the driest section of the rewetted site the seasonal balance increased strongly, but the balance was still negative during the 3 years following rewetting with losses from the system of 44.1, 26.1, 38.3 g CO₂-C m⁻² in 1995, 1996 and 1997 respectively. At the control site seasonal balance during 1995–1997 varied between ecosystem C losses of 41.8 and 95.3 in an area with high Eriophorum cover and between 52.1 and 109.9 g CO₂-C m⁻² with lower cover. Simulation of a cut-away peatland with dense Eriophorum vegetation (Eriophorum cover 70%) showed that if the water level (WT) is low, the seasonal CO₂ balance of the ecosystem can reach the compensation point of no net C change (P _G = R _TOT) only if weather conditions are favourable, but with a high WT the seasonal CO₂ balance would be positive even under varying weather conditions. It can be concluded that with dense Eriophorum vegetation a restored cut-away peatland acts as a functional mire and becomes a sink for atmospheric CO₂. Received: 21 September 1998 / Accepted: 18 May 1999     

Influence of Turn-Taking in a Two-Person Conversation on the Gaze of a Viewer

In natural conversation, the minimal gaps and overlaps of the turns at talk indicate an accurate regulation of the timings of the turn-taking system. Here we studied how the turn-taking affects the gaze of a non-involved viewer of a two-person conversation. The subjects were presented with a video of a conversation while their eye gaze was tracked with an infrared camera. As a control, the video was presented without sound and the sound with still image of the speakers. Turns at talk directed the gaze behaviour of the viewers; the gaze followed, rather than predicted, the speakership change around the turn transition. Both visual and auditory cues presented alone also induced gaze shifts towards the speaking person, although significantly less and later than when the cues of both modalities were available. These results show that the organization of turn-taking has a strong influence on the gaze patterns of even non-involved viewers of the conversation, and that visual and auditory cues are in part redundant in guiding the viewers’ gaze.     

Temporal Fluctuation in North East Baltic Sea Region Cattle Population Revealed by Mitochondrial and Y-Chromosomal DNA Analyses

BackgroundAncient DNA analysis offers a way to detect changes in populations over time. To date, most studies of ancient cattle have focused on their domestication in prehistory, while only a limited number of studies have analysed later periods. Conversely, the genetic structure of modern cattle populations is well known given the undertaking of several molecular and population genetic studies.ResultsBones and teeth from ancient cattle populations from the North-East Baltic Sea region dated to the Prehistoric (Late Bronze and Iron Age, 5 samples), Medieval (14), and Post-Medieval (26) periods were investigated by sequencing 667 base pairs (bp) from the mitochondrial DNA (mtDNA) and 155 bp of intron 19 in the Y-chromosomal UTY gene. Comparison of maternal (mtDNA haplotypes) genetic diversity in ancient cattle (45 samples) with modern cattle populations in Europe and Asia (2094 samples) revealed 30 ancient mtDNA haplotypes, 24 of which were shared with modern breeds, while 6 were unique to the ancient samples. Of seven Y-chromosomal sequences determined from ancient samples, six were Y2 and one Y1 haplotype. Combined data including Swedish samples from the same periods (64 samples) was compared with the occurrence of Y-chromosomal haplotypes in modern cattle (1614 samples).ConclusionsThe diversity of haplogroups was highest in the Prehistoric samples, where many haplotypes were unique. The Medieval and Post-Medieval samples also show a high diversity with new haplotypes. Some of these haplotypes have become frequent in modern breeds in the Nordic Countries and North-Western Russia while other haplotypes have remained in only a few local breeds or seem to have been lost. A temporal shift in Y-chromosomal haplotypes from Y2 to Y1 was detected that corresponds with the appearance of new mtDNA haplotypes in the Medieval and Post-Medieval period. This suggests a replacement of the Prehistoric mtDNA and Y chromosomal haplotypes by new types of cattle.     

Stone Age to recent land-use history at Pegrema, northern Lake Onega, Russian Karelia

The village of Pegrema in Karelia may be regarded as a Stone Age innovation centre in the large Lake Onega area. Two pollen and plant macrofossil diagrams are presented which represent the first contribution to the study of human impact in the area using anthropogenic pollen indicators. A continuous but sporadic human presence from the Mesolithic onwards is demonstrated. While there is no archaeological evidence relating to the period 4200–3000 B.P., the pollen data suggest continuous, though rather sparse human presence. The data do not support any natural catastrophes in Pegrema as has been suggested elsewhere. Cerealia pollen is recorded earlier than expected (c. 5000 B.P.=. In the Bronce Age and Iron Age, the settlement of the Zaonezhye peninsula is reflected by a slight increase in herb pollen representation, sporadic Cerealia pollen and several periods of regression in Picea. The long introductory period of agriculture to the area, as well as the similarities and discrepancies between different sources of evidence (palaeoecological, archaeological and historical) are discussed at some length. The start of land clearance for permanent cultivation in the profile Pegrema S was dated to the late 13^th century. The beginning of more intensive field cultivation in the 15^th century is clearly seen in the pollen succession at both localities. The fluctuation in anthropogenic indicators can be related to population density based on historical data. The village of Pegrema was depopulated in 1956 which is reflected in a distinct decline in settlement indicators. Received February 17 / Accepted May 19, 2000     

Increased expression of matrix metalloproteinase-2 (MMP-2) predicts tumour recurrence and unfavourable outcome in non-small cell lung cancer

The purpose of this study was to analyse the expression of matrix metalloproteinase-2 (MMP-2) and its extracellular matrix metalloproteinase inducer (EMMPRIN) in non-small cell lung cancer (NSCLC), and to evaluate their significance to predict tumour behaviour. The study consists of 212 patients treated by the resection of the tumour. Tumour samples were stained immunohistochemically, and the expression of MMP-2 and EMMPRIN was evaluated both in tumour cells and in peritumoural stromal tissue. The results were compared with clinicopathological factors and survival of the patients. High expression of MMP-2 in tumour cells was found in 83 out of 191 cases (44%). Adenocarcinomas showed more often high expression of MMP-2 as compared with squamous cell or large cell carcinomas (p=0.001). High cancer cell associated MMP-2 expression was associated with increased tumour recurrence (p=0.001). Tumour stroma showed positive staining in 162 (98%) cases and was considered highly stained in 120 (72%) cases. The high stromal MMP-2 expression was noticed more often among large cell carcinomas as compared with other histological types (p=0.007). High cancer cell associated EMMPRIN expression was found in 115 (61%) cases and was associated only with high MMP-2 expression in tumour cells (p=0.006). In overall survival (OS) and disease free survival (DFS) analyses, type of tumour (p=0.001 and p=0.0004), advanced stage (p=0.001 and p=0.013) and high MMP-2 expression in tumour cells (p=0.018 and p=0.001) were associated with poor survival. Also, high stromal MMP-2 expression was related to poor outcome in both OS and DFS analyses (p=0.010 and 0.045, respectively). In multivariate analysis, stromal MMP-2 expression retained its prognostic value to predict OS and DFS (p=0.028 and p=0.039, respectively), together with tumour type and stage (p=0.017, p=0.001 and p=0.021, p=0.008, respectively). The present study shows the significant prognostic value of MMP-2 in NSCLC suggesting that the use of MMP-2 is valuable in determining the patients with more aggressive disease.     

In vitro methods in the prediction of kinetics of drugs: focus on drug metabolism

The absorption, distribution, metabolism, excretion and toxicity (ADMET) properties of a candidate drug influence its final clinical success. These properties have traditionally been evaluated by using various in vivo animal approaches, but recently, a number of in vitro and in silico methods have been introduced to determine key ADMET features. Basic events, such as absorption through the gut wall, binding to plasma proteins, active and passive transfer through the blood-brain barrier, and various metabolic parameters, can now be screened with rapid in vitro and computer modelling methods. The focus in this short review is on the basic in vitro and in silico methods that are used for studying the metabolism properties of new drug molecules.     

Temporospatial expression of matrix metalloproteinases and tissue inhibitors of matrix metalloproteinases in mouse antigen-induced arthritis

Several lines of evidence speak for an important role of matrix metalloproteinases (MMPs) in the development of progressive joint destruction. To better understand the role of MMPs and their tissue inhibitors (TIMPs) in this process, we have used the antigen-induced arthritis model to study the temporospatial expression of several MMPs and TIMPs during the progression of arthritis. Arthritis was induced by a single intra-articular injection of methylated bovine serum albumin (mBSA) into one or both knee joints of adult mice previously immunised against mBSA. Samples were collected at 3, 7, 21 and 42 days after induction of arthritis for histology and RNA extraction, and analysed by Northern hybridisation, histochemistry and immunohistochemistry for production of several MMPs and TIMPs −1, −2 and −3. A systematic analysis of MMP and TIMP mRNA levels in mouse knee joints demonstrated a general upregulation of both MMPs and TIMPs during progression of arthritis. Upregulation of MMP-9, −13 and −14 coincided with the advancement of cartilage degeneration, but the expression patterns of MMP-9 and −13 also followed the course of synovial inflammation. TIMPs were steadily upregulated throughout the examination period. Immunohistochemical localisation of MMPs and TIMPs suggested the synovium to be the major source of MMP and TIMP production in arthritis, although articular cartilage chondrocytes also showed an increased production of both MMPs and TIMPs.