MMP-15 Is Upregulated in Preeclampsia, but Does Not Cleave Endoglin to Produce Soluble Endoglin

Preeclampsia is a major pregnancy complication, characterized by severe endothelial dysfunction, hypertension and maternal end-organ damage. Soluble endoglin is an anti-angiogenic protein released from placenta and thought to play a central role in causing the endothelial dysfunction and maternal organ injury seen in severe preeclampsia. We recently reported MMP-14 was the protease producing placentally-derived soluble endoglin by cleaving full-length endoglin present on the syncytiotrophoblast surface. This find identifies a specific drug target for severe preeclampsia; interfering with MMP-14 mediated cleavage of endoglin could decrease soluble endoglin production, ameliorating clinical disease. However, experimental MMP-14 inhibition alone only partially repressed soluble endoglin production, implying other proteases might have a role in producing soluble endoglin. Here we investigated whether MMP-15–phylogenetically the closest MMP relative to MMP-14 with 66% sequence similarity–also cleaves endoglin to produce soluble endoglin. MMP-15 was localized to the syncytiotrophoblast layer of the placenta, the same site where endoglin was localized. Interestingly, it was significantly (p = 0.03) up-regulated in placentas from severe early-onset preeclamptic pregnancies (n = 8) compared to gestationally matched preterm controls (n = 8). However, siRNA knockdown of MMP-15 yielded no significant decrease of soluble endoglin production from either HUVECs or syncytialised BeWo cells in vitro. Importantly, concurrent siRNA knockdown of both MMP-14 and MMP-15 in HUVECS did not yield further decrease in soluble endoglin production compared to MMP-14 siRNA alone. We conclude MMP-15 is up-regulated in preeclampsia, but does not cleave endoglin to produce soluble endoglin

Fibroid explants reveal a higher sensitivity against MDM2-inhibitor nutlin-3 than matching myometrium

<p>Abstract</p> <p>Background</p> <p>Spontaneous cessation of growth is a frequent finding in uterine fibroids. Increasing evidence suggests an important role of cellular senescence in this growth control. Deciphering the underlying mechanisms of growth control that can be expected not only to shed light on the biology of the tumors but also to identify novel therapeutic targets.</p> <p>Methods</p> <p>We have analyzed uterine leiomyomas and matching normal tissue for the expression of p14^Arfand used explants to see if reducing the MDM2 activity using the small-molecule inhibitor nutlin-3 can induce p53 and activate genes involved in senescence and/or apoptosis. For these studies quantitative real-time RT-PCR, Western blots, and immunohistochemistry were used. Statistical analyses were performed using the student's <it>t </it>test.</p> <p>Results</p> <p>An in depth analysis of 52 fibroids along with matching myometrium from 31 patients revealed in almost all cases a higher expression of p14^Arfin the tumors than in the matching normal tissue. In tissue explants, treatment with the MDM2 inhibitor nutlin-3 induced apoptosis as well as senescence as revealed by a dose-dependent increase of the expression of <it>BAX </it>as well as of <it>p21</it>, respectively. Simultaneously, the expression of the proliferation marker Ki-67 drastically decreased. Western-blot analysis identified an increase of the p53 level as the most likely reason for the increased activity of its downstream markers <it>BAX </it>and <it>p21</it>. Because as a rule fibroids express much higher levels of p14^Arf, a major negative regulator of MDM2, than matching myometrium it was then analyzed if fibroids are more sensitive against nutlin-3 treatment than matching myometrium. We were able to show that in most fibroids analyzed a higher sensibility than that of matching myometrium was noted with a corresponding increase of the p53 immunopositivity of the fibroid samples compared to those from myometrium.</p> <p>Conclusions</p> <p>The results show that uterine fibroids represent a cell population of advanced cellular age compared to matching myometrium. Moreover, the data point to members of the p53-network as to potential novel therapeutic targets for the treatment of uterine fibroids.</p

Identification of Nucleic Acid Binding Sites on Translin-Associated Factor X (TRAX) Protein

Translin and TRAX proteins play roles in very important cellular processes such as DNA recombination, spatial and temporal expression of mRNA, and in siRNA processing. Translin forms a homomeric nucleic acid binding complex and binds to ssDNA and RNA. However, a mutant translin construct that forms homomeric complex lacking nucleic acid binding activity is able to form fully active heteromeric translin-TRAX complex when co-expressed with TRAX. A substantial progress has been made in identifying translin sites that mediate its binding activity, while TRAX was thought not to bind DNA or RNA on its own. We here for the first time demonstrate nucleic acid binding to TRAX by crosslinking radiolabeled ssDNA to heteromeric translin-TRAX complex using UV-laser. The TRAX and translin, photochemically crosslinked with ssDNA, were individually detected on SDS-PAGE. We mutated two motifs in TRAX and translin, designated B2 and B3, to help define the nucleic acid binding sites in the TRAX sequence. The most pronounced effect was observed in the mutants of B3 motif that impaired nucleic acid binding activity of the heteromeric complexes. We suggest that both translin and TRAX are binding competent and contribute to the nucleic acid binding activity

MACSE: Multiple Alignment of Coding SEquences Accounting for Frameshifts and Stop Codons

Until now the most efficient solution to align nucleotide sequences containing open reading frames was to use indirect procedures that align amino acid translation before reporting the inferred gap positions at the codon level. There are two important pitfalls with this approach. Firstly, any premature stop codon impedes using such a strategy. Secondly, each sequence is translated with the same reading frame from beginning to end, so that the presence of a single additional nucleotide leads to both aberrant translation and alignment

Analyses of carnivore microsatellites and their intimate association with tRNA-derived SINEs

BACKGROUND: The popularity of microsatellites has greatly increased in the last decade on account of their many applications. However, little is currently understood about the factors that influence their genesis and distribution among and within species genomes. In this work, we analyzed carnivore microsatellite clones from GenBank to study their association with interspersed repeats and elucidate the role of the latter in microsatellite genesis and distribution. RESULTS: We constructed a comprehensive carnivore microsatellite database comprising 1236 clones from GenBank. Thirty-three species of 11 out of 12 carnivore families were represented, although two distantly related species, the domestic dog and cat, were clearly overrepresented. Of these clones, 330 contained tRNA(Lys)-derived SINEs and 357 contained other interspersed repeats. Our rough estimates of tRNA SINE copies per haploid genome were much higher than published ones. Our results also revealed a distinct juxtaposition of AG and A-rich repeats and tRNA(Lys)-derived SINEs suggesting their coevolution. Both microsatellites arose repeatedly in two regions of the insterspersed repeat. Moreover, microsatellites associated with tRNA(Lys)-derived SINEs showed the highest complexity and less potential instability. CONCLUSION: Our results suggest that tRNA(Lys)-derived SINEs are a significant source for microsatellite generation in carnivores, especially for AG and A-rich repeat motifs. These observations indicate two modes of microsatellite generation: the expansion and variation of pre-existing tandem repeats and the conversion of sequences with high cryptic simplicity into a repeat array; mechanisms which are not specific to tRNA(Lys)-derived SINEs. Microsatellite and interspersed repeat coevolution could also explain different distribution of repeat types among and within species genomes. Finally, due to their higher complexity and lower potential informative content of microsatellites associated with tRNA(Lys)-derived SINEs, we recommend avoiding their use as genetic markers

Health-related physical fitness of adolescents and young adults with myelomeningocele

To assess components of health-related physical fitness in adolescents and young adults with myelomeningocele (MMC), and to study relations between aerobic capacity and other health-related physical fitness components. This cross-sectional study included 50 adolescents and young adults with MMC, aged 16–30 years (25 males). Aerobic capacity was quantified by measuring peak oxygen uptake (peakVO2) during a maximal exercise test on a cycle or arm ergometer depending on the main mode of ambulation. Muscle strength of upper and lower extremity muscles was assessed using a hand-held dynamometer. Regarding flexibility, we assessed mobility of hip, knee and ankle joints. Body composition was assessed by measuring thickness of four skin-folds. Relations were studied using linear regression analyses. Average peakVO2 was 1.48 ± 0.52 l/min, 61% of the participants had subnormal muscle strength, 61% had mobility restrictions in at least one joint and average sum of four skin-folds was 74.8 ± 38.8 mm. PeakVO2 was significantly related to gender, ambulatory status and muscle strength, explaining 55% of its variance. Adolescents and young adults with MMC have poor health-related physical fitness. Gender and ambulatory status are important determinants of peakVO2. In addition, we found a small, but significant relationship between peakVO2 and muscle strength

The Evolution of Compact Binary Star Systems

We review the formation and evolution of compact binary stars consisting of white dwarfs (WDs), neutron stars (NSs), and black holes (BHs). Binary NSs and BHs are thought to be the primary astrophysical sources of gravitational waves (GWs) within the frequency band of ground-based detectors, while compact binaries of WDs are important sources of GWs at lower frequencies to be covered by space interferometers (LISA). Major uncertainties in the current understanding of properties of NSs and BHs most relevant to the GW studies are discussed, including the treatment of the natal kicks which compact stellar remnants acquire during the core collapse of massive stars and the common envelope phase of binary evolution. We discuss the coalescence rates of binary NSs and BHs and prospects for their detections, the formation and evolution of binary WDs and their observational manifestations. Special attention is given to AM CVn-stars -- compact binaries in which the Roche lobe is filled by another WD or a low-mass partially degenerate helium-star, as these stars are thought to be the best LISA verification binary GW sources.Comment: 105 pages, 18 figure

Genome-Wide Analysis of Human Disease Alleles Reveals That Their Locations Are Correlated in Paralogous Proteins

The millions of mutations and polymorphisms that occur in human populations are potential predictors of disease, of our reactions to drugs, of predisposition to microbial infections, and of age-related conditions such as impaired brain and cardiovascular functions. However, predicting the phenotypic consequences and eventual clinical significance of a sequence variant is not an easy task. Computational approaches have found perturbation of conserved amino acids to be a useful criterion for identifying variants likely to have phenotypic consequences. To our knowledge, however, no study to date has explored the potential of variants that occur at homologous positions within paralogous human proteins as a means of identifying polymorphisms with likely phenotypic consequences. In order to investigate the potential of this approach, we have assembled a unique collection of known disease-causing variants from OMIM and the Human Genome Mutation Database (HGMD) and used them to identify and characterize pairs of sequence variants that occur at homologous positions within paralogous human proteins. Our analyses demonstrate that the locations of variants are correlated in paralogous proteins. Moreover, if one member of a variant-pair is disease-causing, its partner is likely to be disease-causing as well. Thus, information about variant-pairs can be used to identify potentially disease-causing variants, extend existing procedures for polymorphism prioritization, and provide a suite of candidates for further diagnostic and therapeutic purposes