Molecular phylogenetic analysis of Tulipa (Liliaceae) based on noncoding plastid and nuclear DNA sequences with an emphasis on Turkey

We investigated the phylogenetic relationships in Tulipa in Turkey using DNA sequences from the plastid trnL‐trnF region and the internal transcribed spacer (ITS) of nuclear ribosomal DNA. We generated trnL‐trnF and nuclear ITS sequences for 11 Tulipa spp. from Turkey and compared the utility of trnL‐trnF and ITS sequences for phylogenetic analysis. Neighbor‐joining, Bayesian and maximum parsimony methods were implemented using the same matrices. Our study of Tulipa based on molecular data revealed congruent results with previous studies. Despite the relatively lower resolution of trnL‐trnF than that of ITS, both sequence matrices generated similar results. Three clades were clearly distinguished, corresponding to subgenera Tulipa, Eriostemones and Orithyia. It is not fully resolved whether Clusianae should be recognized as a separate section of subgenus Tulipa or a distinct subgenus. © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 172 , 270–279.     

Statins inhibit growth of human endometrial stromal cells independently of cholesterol availability

Endometriosis is characterized by ectopic growth of endometrial tissues. Statins, inhibitors of 3-hydroxy-3methylglutaryl-coenzyme A reductase (HMGCR), have been shown to decrease proliferation of several mesenchymal tissues. Actions of statins may be related to decreased availability of cholesterol as well as intermediate metabolites of the mevalonate pathway downstream of HMGCR. This study was designed to evaluate effects of statins on growth of endometrial stromal cells and to investigate mechanisms of these effects. Human endometrial stromal cells were cultured in the absence and in the presence of serum and with or without mevastatin and simvastatin. DNA synthesis and viable cell numbers were determined. Effects of statins were also evaluated in the presence of mevalonate and squalene. Furthermore, effects on phosphorylation of mitogen-activated protein kinase 3/1 (MAPK3/1) (also known as extracellular signal-regulated kinase [ERK1/2]) were determined. Mevastatin and simvastatin induced a concentration-dependent inhibition of DNA synthesis and viable cell count in chemically defined media and in the presence of serum. Mevalonate, but not squalene, abrogated inhibitory effects of statins on cell proliferation. Statins inhibited MAPK3/1 phosphorylation. This is the first study demonstrating that statins inhibit growth of endometrial stromal cells. This effect is also demonstrable in the presence of a supply of cholesterol and may be related to decreased activation of MAPK3/1. The present observations may be relevant to potential therapeutic use of statins in conditions such as endometriosis.     

The diagnostic challenge of progressive pseudorheumatoid dysplasia (PPRD): a review of clinical features, radiographic features, and WISP3 mutations in 63 affected individuals

Progressive pseudorheumatoid dysplasia (PPRD) is a genetic, non-inflammatory arthropathy caused by recessive loss of function mutations in WISP3 (Wnt1-inducible signaling pathway protein 3; MIM 603400), encoding for a signaling protein. The disease is clinically silent at birth and in infancy. It manifests between the age of 3 and 6 years with joint pain and progressive joint stiffness. Affected children are referred to pediatric rheumatologists and orthopedic surgeons; however, signs of inflammation are absent and anti-inflammatory treatment is of little help. Bony enlargement at the interphalangeal joints progresses leading to camptodactyly. Spine involvement develops in late childhood and adolescence leading to short trunk with thoracolumbar kyphosis. Adult height is usually below the 3rd percentile. Radiographic signs are relatively mild. Platyspondyly develops in late childhood and can be the first clue to the diagnosis. Enlargement of the phalangeal metaphyses develops subtly and is usually recognizable by 10 years. The femoral heads are large and the acetabulum forms a distinct "lip" overriding the femoral head. There is a progressive narrowing of all articular spaces as articular cartilage is lost. Medical management of PPRD remains symptomatic and relies on pain medication. Hip joint replacement surgery in early adulthood is effective in reducing pain and maintaining mobility and can be recommended. Subsequent knee joint replacement is a further option. Mutation analysis of WISP3 allowed the confirmation of the diagnosis in 63 out of 64 typical cases in our series. Intronic mutations in WISP3 leading to splicing aberrations can be detected only in cDNA from fibroblasts and therefore a skin biopsy is indicated when genomic analysis fails to reveal mutations in individuals with otherwise typical signs and symptoms. In spite of the first symptoms appearing in early childhood, the diagnosis of PPRD is most often made only in the second decade and affected children often receive unnecessary anti-inflammatory and immunosuppressive treatments. Increasing awareness of PPRD appears to be essential to allow for a timely diagnosis.     

Pro-nerve growth factor induces autocrine stimulation of breast cancer cell invasion through tropomyosin-related kinase A (TrkA) and sortilin protein

The precursor of nerve growth factor (proNGF) has been described as a biologically active polypeptide able to induce apoptosis in neuronal cells, via the neurotrophin receptor p75(NTR) and the sortilin receptor. Herein, it is shown that proNGF is produced and secreted by breast cancer cells, stimulating their invasion. Using Western blotting and mass spectrometry, proNGF was detected in a panel of breast cancer cells as well as in their conditioned media. Immunohistochemical analysis indicated an overproduction of proNGF in breast tumors, when compared with benign and normal breast biopsies, and a relationship to lymph node invasion in ductal carcinomas. Interestingly, siRNA against proNGF induced a decrease of breast cancer cell invasion that was restored by the addition of non-cleavable proNGF. The activation of TrkA, Akt, and Src, but not the MAP kinases, was observed. In addition, the proNGF invasive effect was inhibited by the Trk pharmacological inhibitor K252a, a kinase-dead TrkA, and siRNA against TrkA sortilin, neurotensin, whereas siRNA against p75(NTR) and the MAP kinase inhibitor PD98059 had no impact. These data reveal the existence of an autocrine loop stimulated by proNGF and mediated by TrkA and sortilin, with the activation of Akt and Src, for the stimulation of breast cancer cell invasion.     

A crop tolerating oxidative stress induced by excess lead: maize

Two 14-day-old seedlings of maize (Zea mays L.) cultivars (3223 and Vero) were exposed to different concentrations of lead [0, 2, 5 and 8 mM Pb(NO₃)₂·4H₂O] for 8 days. Exposure of maize cultivars to excess Pb resulted in a significant root growth inhibition though shoot growth and absolute water content remained less affected. The results of chlorophyll a fluorescence indicated that the highly toxic Pb level affected photochemical efficiency in 3223, while no significant effect was observed in the Vero. At the highly toxic Pb concentration, higher membrane leakage was observed in 3223 leaves than that of Vero. This result was related to the accumulation of Pb. On the other hand, the results suggested that there were similar responses in total soluble POD and GR activities with increasing Pb concentrations between both cultivars. But APX activity significantly decreased at highly toxic Pb level in the Vero while a significant increase observed in the 3223. However, SOD activity in 3223 significantly decreased at the highly toxic Pb concentration compared with that at 2 mM Pb concentration. The results of the present study indicated that, Vero withstands excess Pb with its higher Pb accumulation capacity in roots and better upregulated protective mechanisms compared to 3223. Therefore, Vero is more tolerant to Pb toxicity compared to 3223 which was found to be a less tolerant cultivar.     

Modified Vaccinia Virus Ankara Triggers Chemotaxis of Monocytes and Early Respiratory Immigration of Leukocytes by Induction of CCL2 Expression

Orthopoxviruses commonly enter into humans and animals via the respiratory tract. Herein, we show that immigration of leukocytes into the lung is triggered via intranasal infection of mice with modified vaccinia virus Ankara (MVA) and not with the vaccinia virus (VACV) Elstree, Wyeth, or Western Reserve (WR) strain. Immigrating cells were identified as monocytes, neutrophils, and CD4⁺ lymphocytes by flow cytometry and could be detected 24 h and 48 h postinfection. Using an in vitro chemotaxis assay, we confirmed that infection with MVA induces the expression of a soluble chemotactic factor for monocytes, identified as CCL2 (monocyte chemotactic protein-1 [MCP-1]). In contrast to infection with several other VACV strains, MVA induced the expression of CCL2, CCL3, CCL4, and CXCL10 in the human monocytic cell line THP-1 as well as in primary human monocytes. Thus, MVA, and not the VACV Elstree, Wyeth, or WR strain, consistently triggered the expression of a panel of chemokines, including CCL2, in the murine lung, correlating considerably with the immigration of leukocytes. Using CCL2-deficient mice, we demonstrate that CCL2 plays a key role in MVA-triggered respiratory immigration of leukocytes. Moreover, UV irradiation of MVA prevented CCL2 expression in vitro and in vivo as well as respiratory immigration of leukocytes, demonstrating the requirement for an activated molecular viral life cycle. We propose that MVA-triggered chemokine expression causes early immigration of leukocytes to the site of infection, a feature that is important for rapid immunization and its safety and efficiency as a viral vector.The World Health Organization (WHO) announced the worldwide eradication of smallpox at the end of the 1970s. Nevertheless, the threat of an outbreak of smallpox or a smallpox-like disease, either by natural means or via bioterrorism, exists to this day. This danger was illustrated in 2003 by the mini-epidemic of monkeypox in the U.S. Midwest (33). Thus, a vaccine against smallpox is, even today, essential. Although vaccination against smallpox using vaccinia virus (VACV) was quite successful, the incidence of severe side effects prompted the WHO to discontinue the use of the vaccine. Therefore, there is currently still a need for an effective and safe vaccine against smallpox.Among the orthopoxviruses, VACV is frequently used to study poxvirus infection, since it displays many properties of variola virus, the etiologic agent of smallpox, including the capability of modulating and suppressing the immune system by means of expressing several immunoregulatory proteins (44). The assumed natural primary infection site of variola virus is considered to be the respiratory tract (5). Here, the virus encounters lung epithelial cells, conventional dendritic cells, and macrophages. More than three decades ago, viral antigen was detected in alveolar macrophages after a sublethal infection of rabbits with inhaled VACV by using immunofluorescence assays (3). Additionally, under the electron microscope, the cells obtained from repeated washing of rabbit lungs demonstrated that VACV infects and replicates exclusively in macrophages (18). Macrophages, the most abundant hematopoietic cell type in the lung, play a key role in antiviral immune defense through the phagocytosis of infectious particles and the production of reactive oxygen species as well as leukotrienes and inflammatory cytokines (14).The coordinated migration, differentiation, and activation of dendritic cells as well as lymphocytes are required for the efficient elimination of microbes, including viruses, from the lung (15). Activated alveolar macrophages, in particular, enhance the cellular immune response by triggering the immigration of several leukocyte types into the lung owing to the production of chemokines (35). The importance of alveolar macrophages in limiting the replication of a recombinant VACV strain Western Reserve (WR) was recently demonstrated with mice (38). It is likely that the stimulation of cytokine production contributed to the elimination of the virus. In summary, the infection of resident alveolar macrophages with orthopoxviruses and the subsequent upregulation of chemokine expression attract several different leukocyte types, representing a critical event in antiviral defense.A highly attenuated orthopoxvirus strain, modified VACV Ankara (MVA), is being considered as a candidate for the production of a vaccine against smallpox and as a viral vector in gene therapeutic protocols (9). MVA, administered intranasally (i.n.) or intramuscularly as a short-term immunization, has been proven to be protective against a lethal challenge with the virulent VACV strain WR in a mouse model. In contrast, the VACV strain Elstree failed to protect mice when administered intramuscularly 48 h before challenge but was effective when the lethal challenge occurred 14 days postimmunization. Interestingly, an elevated concentration of various types of leukocytes, including monocytes, granulocytes, and T lymphocytes, was present in the lung 48 h after i.n. immunization with MVA (46). This respiratory immigration of leukocytes was most likely triggered by a chemoattractant factor produced by resident lung cells due to infection with MVA. In order to elucidate the mechanism underlying the migration of leukocytes, pilot experiments were performed to test whether MVA triggers the expression of a soluble factor capable of attracting leukocytes, especially monocytes.